![36 Yang et al.
1. Reaction buffer: 10 mM NaH2
PO4
–Na2
HPO4
, pH 7.2.
2. Cu(I) solution: 0.5 mM Cu(CH3
CN)4
PF6
acetonitrile solu-
tion (or Cu(NO3
)2
and TCEP·HCl (Tris[2-carboxyethyl]
phosphine hydrochloride) 1:2 mixture in water, final CuI
concentration 0.5 mM).
1. Solution 1: 40 mg 6-aza-thiothymine (ATT) in 250 mL
HPLC-grade acetonitrile.
2. Solution 2: 1.26 mg spermine in 250 mL autoclaved water
(25 mM).
3. Mix solutions 1 and 2. Centrifuge briefly since some ATT
may not be dissolved.
4. Fucose solution: 8.2 mg fucose dissolved in 1 ml autoclaved
water (50 mM).
1. Cyanogen bromide (5 M in acetonitrile, Sigma–Aldrich)
(Warning: this is a toxic liquid and care should be taken in the
handling of this compound).
2. Reaction buffer: 250 mM morpholineethanesulfonic acid
(MES), pH 7.6, and 20 mM MgCl×6H2
O (see Note 3).
3. Powdered dry ice (see Note 4).
4. Two percent (w/v) solution of lithium perchlorate (LiClO4
)
in acetone (spectral grade, Fisher).
5. Sterilized razor blades.
6. Microcon size-exclusion centrifugal filter devices (Millipore,
YM 10).
1. Exonuclease VII (ExoVII, source: recombinant, Amersham
Biosciences, 1 U converts 1 nmol nucleotide to acid-
soluble nucleotide in 30 min at 37°C under standard assay
conditions).
2. TAMg (1×) buffer.
1. SPI-1 grade highly orderd pyrolytic graphite (HOPG) (SPI
Supplies). Cleave the mica with tape to remove top layer and
create a clean surface for sample deposition.
2. Etched silicon cantilevers (OMCL-AC160TS, Olympus).
A variety of geometrically well-defined single-stranded DNA
templates can be prepared by the following methods. We focus on
the preparation of triangular structures T1 and T2 that have
2.3. Metalation
of DNA Triangle T1
2.4.
MALDI-TOF
2.5.
Chemical Ligation
2.6. Enzymatic
Digestion
of 2D Templates
2.7. Atomic
Force Microscopy
3.
Methods](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-53-320.jpg)
![on Puget-sound, which is from 25 to 30 leagues above Cowlitz.[284]
Besides these stations we visit from time to time, the nearest Forts
where the Catholics in the service of the Hudson Bay Company
reside. This is what takes up almost all our time. We are much in
want of lay brothers and nuns, of school masters and mistresses. We
have to attend to every spiritual as well as temporal affair, which is a
great burden to us. The wives of the Canadians, taken from every
quarter of the country, cause throughout the families a diversity of
languages. They speak almost generally a rude jargon of which we
can scarcely make any use in our public instructions—hence proceed
the obstacles to our progress,—we go along slowly. We are obliged
to teach them French and their catechism together, which occasions
much delay. We are really overwhelmed with business. The savages
apply to us from all sides. Some of them are indifferent, and we
have not time to instruct them. We make them, occasionally, hasty
visits, and baptize the children and the adults who happen to be in
danger of death. But we have no time to learn their languages, and
until now have been without an interpreter to translate the prayers
we wish them to learn. It is only lately that I have succeeded in
translating them into the Tchinoux language. Our difficulties are
greatly increased by this variety of languages; each of the following
tribes has a different dialect: The Kalapouyas, towards the head
waters of the Wallamette,[285] the Tchinoux of the Columbia river;
the Kaijous from Walla-walla; the Pierced Noses, Okanakanes, Flat
Heads, Snakes, Cowlitz, the 231 Klickatates from the interior, north
of Vancouver;[286] the Tcheheles, to the north of the mouth of the
Columbia river; the Nezquales,[287] and those from the interior or of
the Puget sound Bay, those of the Travers river, the Khalams[288] of
the above mentioned bay, those of Vancouver Island, and those
from the northern posts on the sea shore, and from the interior of
the part of the country watered by the tributary streams of the
Travers river, all have their different languages.
Such are the difficulties we have daily to overcome. Our hearts bleed
at the sight of so many souls who are lost under our eyes, without](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-60-320.jpg)
![our being able to carry to them the word of Life. Moreover, our
temporal resources are limited. We are but two, and our trunks did
not arrive last spring by the vessel belonging to the honorable
Hudson Bay Company. We have exhausted our means. The savages,
women and children, ask us in vain for Rosaries. We have no more
Catechisms of the diocese left to distribute among them; no English
Prayer Books for the Catholic Irish; no controversial books to lend.
Heaven appears to be deaf to our prayers, supplications and most
ardent wishes. You can judge of our situation and how much we are
to be pitied. We are in the mean time surrounded by sects who are
using all their efforts to scatter every where the poisonous seeds of
error, and who try to paralyze the little good we may effect.
The Methodists are, first, at Wallamette, which is about eight miles
from my establishment; second, near the Klatraps, south of the
mouth of the Columbia river; third, at Nez Quali, or Puget-sound;
fourth, at the Great Dalles, south of Walla walla; and fifth, at the
Wallamette Falls.[289] The Presbyterian Missions are at Wallawalla,
as you approach Colville.[290] In the midst of so many adversaries
we try to keep our ground firmly; to increase our numbers, 232 and
to visit various parts, particularly where the danger is most pressing.
We also endeavor to anticipate the others, and to inculcate the
Catholic principles in those places where error has not as yet found a
footing, or even to arrest the progress of evil, to dry it up at its
source. The conflict has been violent, but the savages now begin to
open their eyes as to who are the real ministers of Jesus Christ.
Heaven declares itself in our favor. If we had a priest to hold a
permanent station amongst the savages, the country would be ours
in two years. The Methodist Missions are failing rapidly; they are
losing their credit and the little influence they possessed. By the
grace of God, our cause has prevailed at Wallamette. This spring, Mr.
Demers withdrew from the Methodists a whole village of savages,
situate at the foot of the Wallamette Falls. Mr. Demers also visited
the Schinouks [Chinook], below the Columbia river. They are well
disposed towards Catholicity. I have just arrived from Cascades,](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-61-320.jpg)
![which is eighteen leagues from Vancouver. The savages at this place
had resisted all the insinuations of a pretended Minister.[291] It was
my first mission, and only lasted ten days. They learned in that time
the sign of the cross, the offering of their hearts to God, the Lord's
Prayer, the Angelical Salutation, the Apostles' Creed, the ten
Commandments, and those of the Church. I intend to revisit them
soon, near Vancouver, and to baptize a considerable number. Rev.
Mr. Demers has been absent these two months, on a visit to the
savages at the Bay of Puget-sound, who have long since besought
him to come amongst them. I have not been able to visit since the
month of May, my catechumens at Flackimar, a village whose people
were converted last spring, and who had turned a deaf ear to a Mr.
Waller,[292] who is established at Wallamette. Judge then, sir, how
great are our labors, and how much it would advance our 233
mutual interest, were you to send hither one of your Rev. Fathers,
with one of the three lay brothers. In my opinion, it is on this spot
that we must seek to establish our holy religion. It is here that we
should have a college, convent, and schools. It is here that one day
a successor of the Apostles will come from some part of the world to
settle, and provide for the spiritual necessities of this vast region,
which, moreover, promises such an abundant harvest.—Here is the
field of battle, where we must in the first place gain the victory. It is
here that we must establish a beautiful mission. From the lower
stations the Missionaries and Rev. Fathers could go forth in all
directions to supply the distant stations, and announce the word of
God to the infidels still plunged in darkness and the shadows of
death. If your plans should not permit you to change the place of
your establishment, at least take into consideration the need in
which we stand of a Rev. Father, and of a lay brother, to succor us in
our necessities. By the latest dates from the Sandwich Islands, I am
informed that the Rev. Mr. Chochure had arrived there, accompanied
by three priests, the Rev. Mr. Walsh making the fourth.[293] A large
Catholic Church it was hoped would have been ready last autumn for
the celebration of the Holy Mysteries. The natives were embracing](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-62-320.jpg)
![our everlasting faith in great numbers, and the meeting houses were
almost abandoned.
The Bishop of Juliopolis, stationed at Red River,[294] writes to me
that the savages dwelling near the base of the eastern part of the
Rocky Mountains have deputed to him a half blood who resides
amongst them, to obtain from his Grace a priest to instruct them.
Rev. Mr. Thibault is destined for this mission.
I remain, Rev. Father, yours,
F. N. Blanchet.](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-63-320.jpg)
![LETTER XVI
University of St. Louis, 1st Nov. 1842.
Very Rev. Father:
In my last letter of August, I promised to write to you from St. Louis,
should I arrive safely in that city. Heaven has preserved me, and
here I am about to fulfil my promise. Leaving Rev. Father Point and
the Flat Head camp on the river Madison, I was accompanied by
twelve of our Indians. We travelled in three days, a distance of 150
miles, crossing two chains of mountains,[295] in a section of country
frequently visited by the Black Feet warriors, without, however,
meeting with any of these scalping savages. At the mouth of the
Twenty-five Yard River, a branch of the Yellow Stone, we found 250
huts, belonging to several nations, all friendly to us—the Flat Heads,
Kalispels, Pierced Noses, Kayuses, and Snakes. I spent three days
amongst them to exhort them to perseverance, and to make some
preparations for my long journey. The day of my departure, ten
neophytes presented themselves at my lodge to serve as my escort,
and to introduce me to the Crow tribe. On the evening of the second
day we were in the midst of this large and interesting tribe. The
Crows had perceived us from a distance; as we approached, some of
them recognised me, and at the cry of the Blackgown! the
Blackgown! the Crows, young and old, to the number of three
thousand, came out of their wigwams. On entering the village, a
comical scene occurred, of which they suddenly made me the
principal personage. All the chiefs, and 235 about fifty of their
warriors, hastened around me, and I was literally assailed by them.
Holding me by the gown, they drew me in every direction, whilst a
robust savage of gigantic stature, seemed resolved to carry me off
by main force. All spoke at the same time, and appeared to be
quarrelling, whilst I, the sole object of all this contention, could not](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-64-320.jpg)
![wished; three goats, two black-tail deer, a big-horn or mountain
sheep, two fine grey bears, and a swan—to say nothing of the
pheasants, fowls, snipes, ducks and geese.
In the midst of so much game, we scarcely felt the want of bread,
sugar or coffee. The haunches, tongues and ribs replaced these. And
the bed? It is soon arranged. We were in a country where you lose
no time in taking off your shoes; your wrap your buffalo robe around
you, the saddle serves as a pillow, and thanks to the fatigues of a
long journey of about forty miles, under a burning sun, you have
scarcely laid your head upon it before you are asleep. 243 The
gentlemen of Fort Union, at the mouth of the Yellow Stone, received
me with great politeness and kindness. I rested there during three
days. A journey so long and continuous, through regions where the
drought had been so great that every sign of vegetation had
disappeared, had very much exhausted our poor horses. The 1800
miles that we had yet to travel, were not to be undertaken lightly.
After having well considered every thing, I resolved to leave my
horses at the Fort, and to trust myself to the impetuous waters of
the Missouri in a skiff, accompanied by Ignatius and Gabriel. The
result was most fortunate, for, on the third day of our descent, to
our great surprise and joy, we heard the puffing of a steamboat. It
was a real God-send to us; accordingly, our first thought was to
thank God, in all the sincerity of our hearts. We soon beheld her
majestically ascending the stream. It was the first boat that had ever
attempted to ascend the river in that season of the year, laden with
merchandize for the Fur Trade Company. Four gentlemen from New
York, proprietors of the boat, invited me to enter and remain on
board.[296] I accepted with unfeigned gratitude their kind offer of
hospitality; the more so, as they assured me that several parties of
warriors were lying in ambush along the river. On entering the boat I
was an object of great curiosity—my blackgown, my missionary
cross, my long hair, attracted attention. I had thousands of questions
to answer, and many long stories to relate about my journey.](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-71-320.jpg)
![FOOTNOTES:
[1] Volume xxvii of our series begins with chapter xxxiii of the
original New York edition (1838) of Flagg's The Far West. The
author is here describing the part of his journey made in the
late summer or early autumn of 1836.—Ed.
[2] The Vermilion River (which Flagg incorrectly wrote Little
Vermilion) rises, with several branches, in the western and
southern portions of La Salle County, and flows north and west,
entering Illinois River at Rock Island, in Livingston County.
Steelesville (formerly Georgetown) is about fifteen miles east of
Kaskaskia, on the road between Pinkneyville and Chester; the
site was settled on by George Steele in 1810. A block-house fort
erected there in 1812 protected the settlers against attacks
from the Kickapoo Indians. In 1825 a tread-mill was built, and
two years later a store and post-office were erected. The latter
was named Steele's Mills. The settlement was originally called
Georgetown and later changed by an act of state legislature to
Steelesville, being surveyed in 1832.—Ed.
[3] Chester is on the Mississippi River, in Randolph County, just
below the mouth of Kaskaskia River. In the summer of 1829,
Samuel Smith built the first house there, and two years later he,
together with Mather, Lamb and Company, platted the town
site. It was named by Jane Smith from her native town,
Chester, England, and was made the seat of justice for
Randolph in 1848.—Ed.](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-82-320.jpg)
![[4] Flagg is probably referring to Colonel Pierre Menard. See our
volume xxvi, p. 165, note 116.—Ed.
[5] Philadelphia was founded in 1682. There has been much
discussion about the exact date of the founding of Kaskaskia. E.
G. Mason was of the opinion that this uncertainty had arisen in
the confounding of Kaskaskia with an earlier Indian settlement
of the same name on the Illinois River. It seems probable that
Kaskaskia on the Mississippi was started in 1699. Consult E. G.
Mason, Kaskaskia and its Parish Records, in Magazine of
American History (New York, 1881), vi, pp. 161-182, and
Chapters from Illinois History (Chicago, 1901); also C. W.
Alvord, The Old Kaskaskia Records (Chicago Historical Society,
1906). See also A. Michaux's Travels, in our volume iii, p. 69,
note 132.—Ed.
[6] The church of the Immaculate Conception, the first
permanent structure of its kind west of the Alleghany
Mountains, was built in 1720. It was torn down in 1838 and a
large brick church built. For a more detailed description of the
former, see post, pp. 62-64.—Ed.
[7] Hall.—Flagg.
[8] Jacques Marquette was a Jesuit missionary, not a Recollect.
Consult R. G. Thwaites, Father Marquette (New York, 1902). On
Jolliet see Francis Parkman, La Salle (Boston, 1869); and the
latest authority, Ernest Gagnon, Louis Jolliet (Quebec, 1902).—
Ed.
[9] For a short note on the Illinois Indians, consult our volume
xxvi, p. 123, note 86.—Ed.
[10] Flagg errs in saying that Jolliet published an account of his
adventures. His journal was lost in the St. Lawrence River on
the return journey. Father Marquette, however, wrote a journal
of his travels. See Thwaites, Jesuit Relations, lix, which also
contains Jolliet's map of North America (1674).—Ed.](https://image.slidesharecdn.com/1234341-250613075944-8b90956c/85/Dna-Nanotechnology-Methods-And-Protocols-1st-Edition-Chenxiang-Lin-83-320.jpg)
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- 8. DNA Nanotechnology Methods and Protocols Edited by Giampaolo Zuccheri and Bruno Samorì Department of Biochemistry, University of Bologna, Bologna, Italy
- 9. Editors Giampaolo Zuccheri, Ph.D. Department of Biochemistry University of Bologna Bologna, Italy giampaolo.zuccheri@unibo.it Bruno Samorì Department of Biochemistry University of Bologna Bologna, Italy bruno.samori@unibo.it ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-141-3 e-ISBN 978-1-61779-142-0 DOI 10.1007/978-1-61779-142-0 Springer New York Heidelberg London Dordrecht Library of Congress Control Number: 2011929163 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
- 10. v Preface Giorgio Vasari, a painter, architect, and art historian during the Italian Renaissance, is credited with coining the expression “andare a bottega,” (“attending the studio”) refer- ring to the internship that the apprentice would complete in the master’s studio in order to learn what could be uniquely transmitted in person and in that particular environment and that could then lead to making a unique artist of the apprentice. Nowadays, this same concept holds true in science, and despite the many opportuni- ties for communication and “virtual presence”, the real physical permanence in a lab is still the best way for a scientist to learn a technique or a protocol, or a way of thinking. A book of protocols, such as this, humbly proposes itself as the second-best option. Not quite the same as being in person in a lab and witnessing the experts’ execution of a protocol, it still holds many more details and hints than the usually brief methods section found in research papers. This book of protocols for DNA nanotechnology was composed with this concept in mind: prolonging the tradition of Methods in Molecular Biology, it tries to simplify researchers’ lives when they are putting in practice protocols whose results they have learnt in scientific journals. DNA is playing a quite important and dual role in nanotechnology. First, its proper- ties can nowadays be studied with unprecedented detail, thanks to the new instrumental nano(bio)technologies and new insight is being gathered on the biological behavior and function of DNA thanks to new instrumentation, smart experimental design, and proto- cols. Second, the DNA molecule can be decontextualized and “simply” used as a copoly- mer with designed interaction rules. The Watson–Crick pairing code can be harnessed towards implementing the most complicated and elegant molecular self-assembly reported to date. After Ned Seeman’s contribution, elegantly complicated branched structures can be braided and joined towards building nano-objects of practically any desired form. DNA nanotechnology is somewhat like watching professional tennis players: every- thing seems so simple, but then you set foot on the court and realize how difficult it is to hit a nice shot. When you see the structural perfection of a self-assembling DNA nano- object, such as a DNA origami, you marvel at how smart DNA is as a molecule and won- der how many different constructs you could design and realize. Among the others, this book tries to show the procedures to follow in order to repeat some of the methods that lead to such constructs, or to the mastering of the characterization techniques used to study them. Many details and procedures are the fruit of the blending of artistry, science, and patience, which are often unseen in a journal paper, but that could be what makes the difference between a winning shot and hitting the net. Many research groups share their expertise with the readers in this book. For the sake of conciseness, we here mention the group leaders, while it is truly from the daily work of a complete team that the details of a protocol can be worked out. The chapters of this book can be roughly divided into two parts: some deal with the methods of preparing the nanostructures, from the rationale of the operations to the techniques for their handling; some other chapters deal more directly with advanced instrumental techniques that can manipulate and characterize molecules and nanostructures. As part of the first group, Roberto Corradini introduces the reader to the methods and choices for taming helix chirality, Alexander Kotlyar, Wolfgang Fritzsche, Naoki Sugimoto, and James Vesenka
- 11. vi Preface share their different methods in growing, characterizing, and modifying nanowires based on G tetraplexes; Hao Yan and Friedrich Simmel teach all the basics for implementing the self-assembly of branched DNA nanostructures, and then characterizing the assembly. Hanadi Sleiman tells about hybrid metal–DNA nanostructures with controlled geometry. Frank Bier shows the use of rolling circle amplification to make repetitive DNA nanostruc- tures, while, moving closer to technological use of DNA, Arianna Filoramo instructs on how to metalize double-stranded DNA and Andrew Houlton reports on the protocol to grow DNA oligonucleotides on silicon. Also with an eye to the applicative side, Yamuna Krishnan instructs on how to insert and use DNA nanostructures inside living cells. On the instrument side, Ciro Cecconi and Mark Williams introduce the readers to methods for the use of optical tweezers, focusing mainly on the preparation of the ideal molecular construct and on the instrument and its handling, respectively. John van Noort and Sanford Leuba give us protocols on how to obtain sound data from single-molecule FRET and apply it to study the structure of chromatin. Claudio Rivetti teaches the reader how to extract quantitative data from AFM of DNA and its complexes, while Matteo Castronovo instructs on the subtleties of using the AFM as a nanolithography tool on self-assembled monolayers; Jussi Toppari dwelves on the very interesting use of dielectrophoresis as a method to manipulate and confine DNA, while Matteo Palma and Jennifer Cha explain methods for confining on surfaces DNA and those very same types of DNA nanostruc- tures that other chapters tell the reader how to assemble. Aleksei Aksimientev shows the methods for modeling nanopores for implementing DNA translocation, a technique bound to find many applications in the near future. We hope this book will help ignite interest and spur activity in this young research field, expanding our family of enthusiastic followers and practitioners. There are certainly still many chapters to be written on this subject, simply because so much is happening in the labs at this very moment. There will certainly be room for the mainstreaming of pro- tocols on the use of DNA analogues (starting with the marvelous RNA, of course), for the design and preparation of fully 3D architectures, for the development of routes towards functional DNA nanostructures, which will lead to applications. DNA nanostructures can be “re-inserted” in their original biological context, as microorganisms can be convinced to replicate nanostructures or even code them. And eventually, applications will require massive amounts of the nanostructures to be produced and to be manipulated automati- cally, possibly with a precision and output rate similar to that of the assembly of microelec- tronics circuitry nowadays. Our personal wish is that the next chapters will be written by some of our readers. Bologna, Italy Giampaolo Zuccheri Bologna, Italy Bruno Samorì
- 12. vii Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Synthesis and Characterization of Self-Assembled DNA Nanostructures . . . . . . . . 1 Chenxiang Lin, Yonggang Ke, Rahul Chhabra, Jaswinder Sharma, Yan Liu, and Hao Yan 2 Protocols for Self-Assembly and Imaging of DNA Nanostructures . . . . . . . . . . . . 13 Thomas L. Sobey and Friedrich C. Simmel 3 Self-Assembly of Metal-DNA Triangles and DNA Nanotubes with Synthetic Junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Hua Yang, Pik Kwan Lo, Christopher K. McLaughlin, Graham D. Hamblin, Faisal A. Aldaye, and Hanadi F. Sleiman 4 DNA-Templated Pd Conductive Metallic Nanowires . . . . . . . . . . . . . . . . . . . . . . 49 Khoa Nguyen, Stephane Campidelli, and Arianna Filoramo 5 A Method to Map Spatiotemporal pH Changes Inside Living Cells Using a pH-Triggered DNA Nanoswitch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Souvik Modi and Yamuna Krishnan 6 Control of Helical Handedness in DNA and PNA Nanostructures . . . . . . . . . . . . 79 Roberto Corradini, Tullia Tedeschi, Stefano Sforza, Mark M. Green, and Rosangela Marchelli 7 G-Quartet, G-Quadruplex, and G-Wire Regulated by Chemical Stimuli . . . . . . . . 93 Daisuke Miyoshi and Naoki Sugimoto 8 Preparation and Atomic Force Microscopy of Quadruplex DNA . . . . . . . . . . . . . 105 James Vesenka 9 Synthesis of Long DNA-Based Nanowires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Alexander Kotlyar 10 G-Wire Synthesis and Modification with Gold Nanoparticle . . . . . . . . . . . . . . . . . 141 Christian Leiterer, Andrea Csaki, and Wolfgang Fritzsche 11 Preparation of DNA Nanostructures with Repetitive Binding Motifs by Rolling Circle Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Edda Reiß, Ralph Hölzel, and Frank F. Bier 12 Controlled Confinement of DNA at the Nanoscale: Nanofabrication and Surface Bio-Functionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Matteo Palma, Justin J. Abramson, Alon A. Gorodetsky, Colin Nuckolls, Michael P. Sheetz, Shalom J. Wind, and James Hone 13 Templated Assembly of DNA Origami Gold Nanoparticle Arrays on Lithographically Patterned Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Albert M. Hung and Jennifer N. Cha 14 DNA-Modified Single Crystal and Nanoporous Silicon . . . . . . . . . . . . . . . . . . . . 199 Andrew Houlton, Bernard A. Connolly, Andrew R. Pike, and Benjamin R. Horrocks
- 13. viii Contents 15 The Atomic Force Microscopy as a Lithographic Tool: Nanografting of DNA Nanostructures for Biosensing Applications . . . . . . . . . . . . . . . . . . . . . . 209 Matteo Castronovo and Denis Scaini 16 Trapping and Immobilization of DNA Molecules Between Nanoelectrodes . . . . . 223 Anton Kuzyk, J. Jussi Toppari, and Päivi Törmä 17 DNA Contour Length Measurements as a Tool for the Structural Analysis of DNA and Nucleoprotein Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Claudio Rivetti 18 DNA Molecular Handles for Single-Molecule Protein-Folding Studies by Optical Tweezers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Ciro Cecconi, Elizabeth A. Shank, Susan Marqusee, and Carlos Bustamante 19 Optimal Practices for Surface-Tethered Single Molecule Total Internal Reflection Fluorescence Resonance Energy Transfer Analysis . . . . . . . . . . . . . . . . 273 Matt V. Fagerburg and Sanford H. Leuba 20 Engineering Mononucleosomes for Single-Pair FRET Experiments . . . . . . . . . . . 291 Wiepke J.A. Koopmans, Ruth Buning, and John van Noort 21 Measuring DNA–Protein Binding Affinity on a Single Molecule Using Optical Tweezers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 Micah J. McCauley and Mark C. Williams 22 Modeling Nanopores for Sequencing DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 Jeffrey R. Comer, David B. Wells, and Aleksei Aksimentiev Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
- 14. ix Contributors Justin J. Abramson • Department of Mechanical Engineering, Columbia University, New York, NY, USA Aleksei Aksimentiev • Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA Faisal A. Aldaye • Department of Systems Biology, Harvard Medical School, Boston, MA, USA Frank F. Bier • Department of Nanobiotechnology & Nanomedicine, Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany Ruth Buning • Leiden Institute of Physics, Leiden Universiteit, Leiden, The Netherlands Carlos Bustamante • Howard Hughes Medical Institute, Department of Physics, University of California, Berkeley, CA, USA Stephane Campidelli • CEA Saclay, Laboratoire d’Electronique Moléculaire, Gif-sur-Yvette Cedex, France Matteo Castronovo • Department of Biology, MONALISA Laboratory, College of Science and Technology, Temple University, PA, USA Ciro Cecconi • CNR-Istituto Nanoscienze S3, Department of Physics, University of Modena e Reggio Emilia, Modena, Italy Jennifer N. Cha • Department of Nanoengineering, UC San Diego, La Jolla, CA, USA Rahul Chhabra • University of Alberta, National Institute of Nanotechnology, Edmonton, AB, Canada Jeffrey R. Comer • Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA Bernard A. Connolly • Chemical Nanoscience Laboratory, School of Chemistry, Newcastle University, Newcastle upon Tyne, UK Roberto Corradini • Dipartimento di Chimica Organica e Industriale, Univeristà di Parma, Parma, Italy Andrea Csaki • Institute of Photonic Technology (IPHT), Jena, Germany Matt V. Fagerburg • Departments of Cell Biology and Physiology and Bioengineering, University of Pittsburgh School of Medicine and Swanson School of Engineering, Petersen Institute of Nano Science and Engineering and University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA Arianna Filoramo • CEA Saclay, Laboratoire d’Electronique Moléculaire, Gif-sur-Yvette Cedex, France Wolfgang Fritzsche • Institute of Photonic Technology (IPHT), Jena, Germany Alon A. Gorodetsky • Department of Chemistry, Columbia University, New York, NY, USA
- 15. x Contributors Mark M. Green • Dipartimento di Chimica Organica e Industriale, Univeristã di Parma, Parma, Italy Graham D. Hamblin • Department of Chemistry, McGill University, Montreal, Canada Ralph Hölzel • Department of Nanobiotechnology & Nanomedicine, Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany James Hone • Department of Mechanical Engineering, Columbia University, New York, NY, USA Benjamin R. Horrocks • Chemical Nanoscience Laboratory, School of Chemistry, Newcastle University, Newcastle upon Tyne, UK Andrew Houlton • Chemical Nanoscience Laboratory, School of Chemistry, Newcastle University, Newcastle upon Tyne, UK Albert M. Hung • Department of Nanoengineering, UC San Diego, La Jolla, CA, USA Yonggang Ke • Dana-Farber Cancer Institute & Harvard Medical School, Boston, MA, USA Wiepke J.A. Koopmans • Leiden Institute of Physics, Leiden Universiteit, The Netherlands Alexander Kotlyar • Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel Yamuna Krishnan • Biochemistry, Biophysics and Bioinformatics, National Centre for Biological Sciences, Bangalore, India Anton Kuzyk • Lehrstuhl für Bioelektronik, Physik-Department and ZNN/WSI, Technische Universität München, Garching, Germany Christian Leiterer • Institute of Photonic Technology (IPHT), Jena, Germany Sanford H. Leuba • Departments of Cell Biology and Physiology and Bioengineering, University of Pittsburgh School of Medicine and Swanson School of Engineering, Petersen Institute of NanoScience and Engineering, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA Chenxiang Lin • Dana-Farber Cancer Institute & Wyss Institute at Harvard University, Boston, MA, USA Yan Liu • Department of Chemistry and Biochemistry, The Biodesign Institute, Arizona State University, Tempe, AZ, USA Pik Kwan Lo • Department of Chemistry, McGill University, Montreal, Canada Rosangela Marchelli • Dipartimento di Chimica Organica e Industriale, Univeristà di Parma, Parma, Italy Susan Marqusee • Department of Molecular & Cell Biology, University of California, Berkeley, CA, USA Micah J. McCauley • Department of Physics, Northeastern University, Boston, MA, USA Christopher K. McLaughlin • Department of Chemistry, McGill University, Montreal, Canada Daisuke Miyoshi • Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), and Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, Japan
- 16. xi Contributors Souvik Modi • Biochemistry, Biophysics and Bioinformatics, National Centre for Biological Sciences, Bangalore, India Khoa Nguyen • CEA Saclay, Laboratoire d’Electronique Moléculaire, Gif-sur-Yvette Cedex, France Colin Nuckolls • Department of Chemistry, Columbia University, New York, NY, USA Matteo Palma • Department of Mechanical Engineering & Applied Physics and Applied Mathematics, Columbia University, New York, NY, USA Andrew R. Pike • Chemical Nanoscience Laboratory, School of Chemistry, Newcastle University, Newcastle upon Tyne, UK Edda Reiß • Department of Nanobiotechnology & Nanomedicine, Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Potsdam, Germany Claudio Rivetti • Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy Denis Scaini • Sincrotrone Trieste, Basovizza, Trieste, Italy Stefano Sforza • Dipartimento di Chimica Organica e Industriale, Univeristà di Parma, Parma, Italy Elizabeth A. Shank • Harvard Medical School, Boston, MA, USA Jaswinder Sharma • Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, NM, USA Michael P. Sheetz • Department of Biological Sciences, Columbia University, New York, NY, USA Friedrich C. Simmel • Physik Department, Technische Universität München, Munich, Germany Hanadi F. Sleiman • Department of Chemistry, McGill University, Montreal, Canada Thomas L. Sobey • Physik Department, Technische Universität München, Munich, Germany Naoki Sugimoto • Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), and Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, Japan Tullia Tedeschi • Dipartimento di Chimica Organica e Industriale, Università di Parma, Parma, Italy J. Jussi Toppari • Department of Physics, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland Päivi Törmä • Department of Applied Physics, School of science, Aalto University, Aalto, Finland John van Noort • Leiden Institute of Physics, Leiden Universiteit, Leiden, The Netherlands James Vesenka • Department of Chemistry and Physics, University of New England, Biddeford, ME, USA David B. Wells • Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA
- 17. xii Contributors Mark C. Williams • Department of Physics, Northeastern University, Boston, MA, USA Shalom J. Wind • Department of Applied Physics and Applied Mathematics, Columbia University, New York, NY, USA Hao Yan • Department of Chemistry and Biochemistry, The Biodesign Institute, Arizona State University, Tempe, AZ, USA Hua Yang • Department of Chemistry, University of British Columbia, Vancouver, Canada
- 18. 1 Giampaolo Zuccheri and Bruno Samorì (eds.), DNA Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 749, DOI 10.1007/978-1-61779-142-0_1, © Springer Science+Business Media, LLC 2011 Chapter 1 Synthesis and Characterization of Self-Assembled DNA Nanostructures Chenxiang Lin, Yonggang Ke, Rahul Chhabra, Jaswinder Sharma, Yan Liu, and Hao Yan Abstract The past decade witnessed the fast evolvement of structural DNA nanotechnology, which uses DNA as blueprint and building material to construct artificial nanostructures. Using branched DNA as the main building block (also known as a “tile”) and cohesive single-stranded DNA (ssDNA) ends to designate the pairing strategy for tile–tile recognition, one can rationally design and assemble complicated nanoarchi- tectures from specifically designed DNA oligonucleotides. Objects in both two- and three-dimensions with a large variety of geometries and topologies have been built from DNA with excellent yield; this development enables the construction of DNA-based nanodevices and DNA-template directed organiza- tion of other molecular species. The construction of such nanoscale objects constitutes the basis of DNA nanotechnology. This chapter describes the protocol for the preparation of ssDNA as starting material, the self-assembly of DNA nanostructures, and some of the most commonly used methods to characterize the self-assembled DNA nanostructures. Key words: DNA nanotechnology, Self-assembly, Electrophoresis, Atomic force microscopy The notion that DNA is merely the gene encoder of living systems has been eclipsed by the successful development of DNA nano- technology. DNA is an excellent nanoconstruction material because of its inherent merits: First, the rigorous Watson-Crick base-pairing makes the hybridization between DNA strands highly predictable. Second, the structure of the B-form DNA double helix is well-understood; its diameter and helical repeat have been determined to be ~2 and ~3.4 nm (i.e., ~10.5 bases), respectively, which facilitates the modeling of even the most com- plicated DNA nanostructures. Third, DNA possesses combined 1. Introduction
- 19. 2 Lin et al. structural stiffness and flexibility. The rigid DNA double helixes can be linked by relatively flexible single-stranded DNA (ssDNA) to build stable motifs with desired geometry. Fourth, modern organic chemistry and molecular biology have created a rich tool- box to readily synthesize, modify, and replicate DNA molecules. Finally, DNA is a biocompatible material, making it suitable for the construction of multicomponent nanostructures made from hetero-biomaterials. The field of structural DNA nanotechnology began with Nadrian Seeman’s vision of combining branched DNA molecules bearing complementary sticky-ends to construct two-dimensional (2D) arrays (1) and his experimental construction of a DNA object topologically equal to a cube (2). Today, DNA self-assembly has matured with such vigor that it is currently possible to build micro- or even millimeter-sized nanoarrays with desired tile geometry and periodicity as well as any discrete 2D or 3D nano- structures we could imagine (3–8). Modified by functional groups, those DNA nanostructures can serve as scaffolds to con- trol the positioning of other molecular species (9–21), which opens opportunities to study intermolecular synergies, such as protein–protein interactions, as well as to build artificial multi- component nanomachines (22–24). Generally speaking, the creation of a novel DNA motif usu- ally requires the following steps: (1) Structural modeling: physical and/or graphic models are used to help the design of a new DNA motif; (2) Sequence design: in this step, specific sequences are assigned to all ssDNA molecules in the model; (3) Experimental synthesis of the DNA nanostructure; and (4) Characterization of the DNA nanostructure. The first two steps are crucial to pro- gram the outcome of self-assembly and assisted by computer soft- ware (25–30). In this chapter, we are going to describe the experimental protocols involved in steps 3 and 4. All chemicals are purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. All buffer solutions are filtered and stored at room temperature unless otherwise noted. 1. Synthetic ssDNA (Integrated DNA Techonologies, Coralville, IA) with designated sequences. 2. TBE buffer (1×): 89 mM Tris–boric acid, pH 8.0, 2 mM eth- ylenediaminetetraacetic acid disodium salt (EDTA-Na2 ). 3. 20%urea-acrylamideMix:20%acrylamide(19:1acrylamide:bis, Bio-Rad Laboratories, Hercules, CA), 8.3 M urea in 1× TBE buffer. 2. Material 2.1. Denaturing Polyacrylamide Gel Electrophoresis for the Purification of Synthetic Single- Stranded DNA
- 20. 3 Synthesis and Characterization of Self-Assembled DNA Nanostructures 4. 0% Urea-acrylamide Mix: 8.3 M Urea in 1× TBE buffer. 5. Ammonium persulfate (APS): prepare 10% water solution and store at 4ºC. 6. N,N,N,N¢-tetramethyl-ethylenediamine (TEMED, Bio-Rad). 7. Bromophenol blue (BB) or xylene cyanole FF (XC) (2×): prepare 0.1% w/v solution of the dye in 90% formamide solu- tion containing 10 mM NaOH and 1 mM Na2 EDTA. 8. Ethidium bromide: prepare 300 mL 0.1 mg/mL solution in a glass tray for gel staining. 9. Elution buffer (1×): 500 mM ammonium acetate, 10 mM magnesium acetate, 2 mM EDTA-Na2 . 10. 1-Butanol and 100% Ethanol. 11. Spin X centrifuge tube filters (Corning, Lowell, MA). 1. Polyacrylamide Gel Electrophoresis (PAGE) purified ssDNA. 2. TAE-Mg buffer (10×): 0.4 M Tris–acetic acid, pH 8.0, 125 mM magnesium acetate, 20 mM EDTA-Na2 . 1. Self-assembled DNA nanostructures. 2. 40% acrylamide (19:1 acrylamide:bis, Bio-Rad Laboratories, Hercules, CA) solution. 3. Non-denaturing loading buffer (10×): 0.2% w/v bromophe- nol blue and xylene cyanole FF in 1× TAE-Mg buffer con- taining 50% v/v glycerol. 4. DNA ladder with suitable size (Invitrogen, Carlsbad, CA). 5. TAE-Mg buffer (1×), TEMED, and 10% APS solution (vide supra). 6. Stains-All: prepare 0.01% w/v Stains-All in 45% v/v forma- mide solution. 1. Self-assembled DNA nanostructures. 2. TAE-Mg buffer (1×) (vide supra). 3. Mica discs (Ted Pella, Inc) and Atomic Force Microscope (AFM) cantilevers of choice with integrated probes (such as NP-S from Veeco, Inc for imaging in liquids). With advanced solid state synthesis chemistry, DNA synthesizer can generate DNA strands with designated sequences up to 200- base long. However, a significant yield drop is normally associ- ated with the synthesis of longer DNA strands. For example, 2.2. Self-Assembly of DNA Nanostructures 2.3. Non-denaturing PAGE for the Characterization of Self-Assembled DNA Nanostructures 2.4. Atomic Force Microscope Imaging of Self-Assembled DNA Arrays 3. Methods 3.1. Denaturing PAGE Purification of Synthetic ssDNA
- 21. 4 Lin et al. if the yield for the addition of one nucleoside is 99%, the yield for the synthesis of a 100-mer ssDNA is only ~37%. Therefore, it is very important to purify the synthetic DNA strands that are lon- ger than 30 bases to maximize the self-assembly yield in the next step. Effective ways to purify ssDNA less than 200-base long include high performance liquid chromatography (HPLC) and PAGE. Here, we discuss the protocol for denaturing PAGE puri- fication of synthetic DNA strands. 1. Set up the gel assembly following the manufacturer’s instruc- tion (we use a Hofer SE 600 Ruby from GE Healthcare) (see Note 1). 2. Mix proper volume of 20% and 0% Urea-acrylamide stock solution to prepare the acrylamide solution with desired con- centration. Each gel needs ~35 mL acrylamide solution. For example, to make an 8% polyacrylamide gel, take 14 mL of 20% Urea-acrylamide stock and mix with 21 mL of 0% Urea- acrylamide stock. Stir thoroughly to mix well. For each gel, add 262 mL of 10% APS solution and 14.7 mL of TEMED. Stir thoroughly to mix well. 3. Quickly cast the gel using 35 mL pipette and insert the comb. Make sure no air bubble is trapped in the gel. Leave the gel at room temperature for at least 30 min to allow it solidifies. 4. Prepare the DNA sample. Add DI water to each dry samples to make 0.5 OD260 /mL DNA solution. Take 4 OD of each sample (8 mL) into newly labeled tubes (see Note 2) and the rest of the samples should be stored at −20ºC. Add 2× dena- turing dye to each sample (BB, XC, or both) and add water to adjust the final volume to 20 mL. Heat the sample at 90ºC for 5 min to denature the DNA strands (see Note 3). 5. When the gel has polymerized, remove the combs and attach the upper buffer chamber (UBC) to the gel assembly. Add 1×TBE buffer (running buffer) to the UBC and rinse the wells thoroughly with glass pipette. Drain the UBC and add fresh running buffer to cover all the wells. 6. Load the samples to each well. Load 10 mL/well into the gel wells (generally 2 OD per lane) using the gel loading tips. Be careful not to flush the sample out of the well (see Note 4). 7. Carefully put the UBC and gel assembly into the lower buffer chamber (LBC) with ~3.5 L 1× TBE buffer. Add buffers into both UBC and LBC to the marked MAX lines (see Note 5). 8. Turn on the circulating water and set the temperature to 35ºC. Secure the lid of the gel box and connect the electrodes to a DC power supply. Make sure the polarity is correct. Run gel at constant current ~30–40 mA per gel for around 2–3 h depending on the length of the interested DNA fragments.
- 22. 5 Synthesis and Characterization of Self-Assembled DNA Nanostructures The tracking dye in the loading buffer provides a rough marker of the migration of DNA fragments (Table 1). 9. Turn off the power supply and circulating water. Lift the gel from the gel assembly and carefully put it into a glass try con- taining ~300 mL ethidium bromide (see Note 6). Stain the gel for 5 min and destain it for 5 min in distilled water. 10. In a dark room, lift the gel gently and put it on the UV transil- luminator. Turn on the UV at wavelength of 302 nm, use razor blade to cut the major band out (see Note 7). Turn off the UV lamp, chop the band into small pieces, and collect the small gel blocks into Spin X centrifuge tube filters. Add 500 mL of elution buffer into each filter; shake in cold room (4ºC) overnight before proceed to the next step (see Note 8). 11. Centrifuge the Spin X tube filters (4,600×g for 6 min) to separate the elution buffer from gel blocks. Add 1 mL of 1-butanol to the collected elution buffer, vortex the tube for 1 min, and centrifuge it at 600×g for 1 min. After the spin, discard the upper layer of 1-butanol with pipette into waste bottle under venting hood. The 1-butanol washing extracts ethidium bromide and tracking dyes from the DNA sample. 12. Add in 1 mL ethanol to the DNA sample and mix well. Leave the mixture in −20ºC freezer for 30 min. Spin at 16,200×g for 30 min at 4ºC to precipitate DNA. Pour out the ethanol and wash the DNA pellet with 70% v/v ice cold ethanol if desired. Centrifuge the tube at 16,200×g for 10 min after ethanol washing and pour out all liquid. 13. Use a vacuum concentrator (we use a Vacufuge from Eppendorf, Westbury, NY) to dry the purified DNA sample for 1 h at 30ºC. Add in 50 mL distilled H2 O, vortex for 1 min to dissolve the DNA sample. Measure the absorbance of the DNA solution at 260 nm (OD260 ) using a UV-Vis spectrom- eter (we employ a Biophotometer from Eppendorf) and con- vert the measured OD260 value to molar concentration using Table 1 The tracking dye migration on polyacrylamide denaturing gels (Dyes migrate to the same point as DNA strand of the indicated size in a denaturing polyacrylamide gel) Polyacrylamide concentration 5% 6% 8% 10% 12% Bromophenol blue (bp) 35 26 19 12 8 Xylene cyanole FF (bp) 130 106 76 55 26
- 23. 6 Lin et al. the extinction coefficient (e) of the DNA strand provided by the oligonucleotide vendor. Adjust the concentration of all purified DNA strand solution to 30 mM (or any other value to the experimenter’s convenience) by adding distilled H2 O. Store all DNA samples in −20ºC freezer. The formation of hydrogen bonded DNA complex is a self- assembly process. The DNA strands are mixed at stoichiometric molar ratio in a near-neutral buffer containing divalent cations (usually Mg2+ ), heated to denature and then gradually cooled to allow the ssDNA molecules to find their correct partners and adopt the most energy-favorable conformation. 1. Add stoichiometric amount of DNA strands into one 1.5 mL tube (or any other suitable tube size). Add 10× TAE-Mg buf- fer and distilled H2 O to adjust the final concentration of each DNA strand to be 1 mM or any other desired concentration. Mix well and close the tube tightly. 2. This mixture is then heated on a heat block to 95ºC for 5 min and cooled to the desired temperature by the following pro- tocol: 20 min at 65ºC, 20 min at 50ºC, 20 min at 37ºC, and if desired, 20 min at room temperature. 3. To assemble large DNA constructs, such as 2D arrays, slow annealing is desirable. In this case, the mixture is placed on a floating rack, transferred to a 2 L water bath, which is pre- heated to about 90°C and placed inside a Styrofoam box, and allowed to cool slowly to the desired temperature over the period of 2 days. This slow annealing process can also be car- ried out on a thermal cycler (see Note 9). Non-denaturing PAGE is an effective assay to characterize the self-assembled DNA supermolecules. Well-formed DNA nano- structure should migrate as a distinct band after electrophoresis. Non-denaturing PAGE also provides information regarding the yield of self-assembly. A typical gel image showing the correct formation of four helix DNA tile (31) is shown in Fig. 1. 1. Set up the gel assembly following the manufacturer’s instruc- tion as described in step 1, Subheading 3.1 (we use a Hoefer SE 600 Ruby, GE Healthcare). 2. Prepare non-denaturing acrylamide mixture from 40% acryl- amide (acrylamide:bis 19:1) stock, 10× TAE-Mg buffer and distilled H2 O. The final mixture should contain 1× TAE-Mg buffer. For example, to make an 8% non-denaturing gel, mix 7 mL of 40% acrylamide stock, 3.5 mL of 10× TAE-Mg buf- fer, and 24.5 mL H2 O. Stir thoroughly to mix well. For each gel, add 262 mL of 10% APS solution and 14.7 mL of TEMED. Stir thoroughly to mix well. 3.2. Anneal DNA Strands to Self- Assemble DNA Nanostructures 3.3. Non-denaturing PAGE for the Characterization of Self-Assembled DNA Nanostructures
- 24. 7 Synthesis and Characterization of Self-Assembled DNA Nanostructures 3. Quickly cast the gel using 35 mL pipette and insert the comb. Make sure no air bubble is trapped in the gel. Leave the gel at room temperature for at least 2 h to allow it solidify (see Note 10). 4. When the gel has polymerized, remove the combs and attach the UBC to the gel assembly. Add 1× TAE-Mg buffer (run- ning buffer) to the UBC and rinse the wells thoroughly with glass pipette. Drain the UBC and add fresh running buffer to cover all the wells. 5. Add 10× non-denaturing loading buffer to the preannealed DNA samples (finally, the DNA should be in 1× loading buf- fer). Vortex to mix well. Immediately load the DNA samples to each well using the gel loading tips. (Be careful not to flush the sample out of the well.) Take note about the sequence of the samples loaded. A DNA ladder with proper size should be loaded into a separate lane as a reference. 6. Immerse the gel assembly (together with UBC) to the 1× TAE-Mg buffer in the LBC. Add buffers into both UBC and LBC to the marked MAX lines. It is important not to disturb the sample when adding buffer to UBC. Add buffer gently along the side of the chamber. 7. Turn on the circulating water and set the temperature to 20ºC. Secure the lid of the gel box and connect the elec- trodes to a DC power supply. Make sure the polarity is cor- rect. Run gel at constant voltage ~200 V for 4–8 h depending on the size of the interested DNA complexes. Fig.1. Nondenaturing gel (8% polyacrylamide) of the 4-helix complex stained with Stains-All. Equimolar mixtures of 1 mM of each strand were annealed, and the electro- phoresis was run at room temperature. Lane M is a 100 bp DNA ladder. Lanes 1–8 contain complexes with partial combination of the component strands. Strands included in the annealing are indicated with a schematic drawing above the lane. Lane 9 corre- sponds to the full complex with all of the component strands.
- 25. 8 Lin et al. 8. Turn off the power supply and circulating water. Lift the gel from the gel assembly and carefully put it into a glass tray con- taining ~300 mL 0.01% Stains-All solution. (Wear gloves all time when working with Stains-All). Low percentage gels can be fragile thus should be treated with extreme care. Stain the gel for 2 h and then rinse the gel well using distilled H2 O. 9. Place a slide of transparency on the white lamp of the translu- minator, carefully put the gel on it, and turn on white light to destain the gel until the gel appears almost colorless. This takes approximately 10–20 min. Watch the color change to avoid overdestain. 10. Use Kimwipe to wick the water off as much as possible, and then cover the gel with another transparency. Make sure no air bubble is trapped between the gel and the transparency. Also avoid stripes and pattern caused by thin layer of water. Scan the gel on a desktop scanner and save the image. 1. The protocol described here assumes the use of PicoPlus AFM (Agilent). To start the imaging session, turn on the computer, Pico Scan controller, and then the AC controller. Open software “Pico Scan.” 2. Choose tapping mode AFM (AC AFM) and insert proper AFM tip into the tip holder on the top of the scanner. For AAC (acoustic AC) mode, use the gold-coated silicon nitride tip (NP-S tip, Veeco) for imaging in liquid or the proper acoustic AC tip (Veeco) for imaging in air. For the NP-S tips, use the tip on the thinner and shorter cantilever for imaging. 3. Sample preparation: Assemble a piece of freshly cleaved mica as the bottom of the fluid cell on the sample stage. Spot a 2 mL of 1 mM NiCl2 solution on mica and leave it to adsorb on the surface for 2 min. Then, add a 2 mL of the sample to the spot and leave it to adsorb on the surface for another 2 min. Finally, add 400 mL 1× TAE-Mg buffer onto the mica in the fluid cell. The Ni2+ adsorbed on mica surface can help the DNA array stay on the surface during the scanning. Attach the sample stage to the magnetic posts on the AFM. 4. Place the scanner on the sample stage with the tip pointing down. Lock the scanner. Turn on laser switch and plug in the detector. Move the laser spot so that it is on the back of the cantilever tip. Adjust the position of the photodiode inside the detector to maximize the sum of signal. Also make sure that the reflected laser spot is at the center of the photodiode. 5. Tune the tip and choose drive frequency with maximum amplitude. Set the parameters for scanning. Proportional gain and integral gain are 0.5 both or larger (<1.2). Use the servo range of ~4,180 nm. Set 0.85 for the amplitude set point. 3.4. AFM Imaging of Self-Assembled DNA Arrays
- 26. 9 Synthesis and Characterization of Self-Assembled DNA Nanostructures Start approaching the tip to the surface and wait until the servo is active. The approaching process may take a few minutes if the tip was far from the surface initially. 6. When servo is active, start scanning. During the scanning, optimize the parameters (gains, amplitude set point, and servo range) to obtain optimal images. Scanning size depends on the size and morphology of the DNA assembly of interest. Zoom in on area of interest to observe the detail of the DNA nanostructures. Save the images on the computer. A typical AFM image of the DNA 2D arrays self-assembled from eight helix tiles (31) is shown in Fig. 2. 7. To end the imaging session, stop scanning and withdraw the tip from the surface. Take out the sample stage and dissemble the mica. Remove the tip from the scanner. Clean the sample stage, mica, and scanner head for future use. 1. It is crucial to clean all parts of the gel assembly, especially the glass plates and combs. The glass plates can be cleaned by rinsing extensively with distilled water followed by ethanol and acetone. Also check the edges of glass plates; they should be free of indentation. 2. Each lane of the gel can hold 2 OD of DNA; to purify larger amount, simply take more sample and lanes. 4. Notes Fig. 2. AFM images of DNA arrays self-assembled from eight helix tiles. Image sizes are 4.5×4.5 mm2 on the left and 800×800 nm2 on the right. Each square on the zoom-in image represents an eight helix tile as shown in schematic at the bottom right corner.
- 27. 10 Lin et al. 3. Do not heat the sample longer than 10 min. Immediately place the heated tubes on ice to better denature DNA strands with complex secondary structures. 4. Take note about the sequence of the samples loaded. Leave one lane empty in between different samples, especially when their lengths (bp) are very close. The samples should not be loaded too slow to prevent diffusion, which may lead to band smearing. 5. It is important not to disturb the sample when adding buffer to UBC. Add buffer gently along the side of the chamber. 6. Wear nitrile gloves all time when working with ethidium bro- mide. Low percentage gels can be fragile thus should be treated with extreme care. 7. The major band may show darker than the surrounding because the UV absorbance of DNA is so high, especially when more than 2 OD of DNA is loaded in each lane. 8. To optimize the elution yield, freeze small blocks of gel at −20ºC for 10 min before adding elution buffer. This is espe- cially helpful when purifying DNA strands longer than 100 bases. 9. The annealed structures should be handled gently (e.g., do not vortex) and stored at 4ºC. 10. Low percentage gels can take longer time to solidify. Always make sure that the gel solidifies completely before removing the comb. Acknowledgments This work was supported by grants from the National Science Foundation (NSF), the Army Research Office (ARO), and the Technology and Research Initiative Fund from Arizona State University to Y.L. and by grants from NSF, ARO, Air Force Office of Scientific Research, Office of Naval Research, and the National Institute of Health to H.Y. References 1. Seeman, N. C. (1982) Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247. 2. Chen J., and Seeman, N. C. (1991) The synthesis from DNA of a molecule with the connectivity of a cube. Nature 350, 631–633. 3. Seeman, N. C. (2003) DNA in a material world. Nature 421, 427–431. 4. Deng, Z. X., Lee, S. H., and Mao, C. D. (2005) DNA as nanoscale building blocks. J. Nanosci. Nanotechnol. 5, 1954–1963. 5. Turberfield, A. J. (2003) DNA as an engineer- ing material. Phys. World 16, 43–46. 6. Lin, C., Liu, Y., Rinker, S., and Yan, H. (2006) DNA Tile based self-assembly: building
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- 30. 13 Giampaolo Zuccheri and Bruno Samorì (eds.), DNA Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 749, DOI 10.1007/978-1-61779-142-0_2, © Springer Science+Business Media, LLC 2011 Chapter 2 Protocols for Self-Assembly and Imaging of DNA Nanostructures Thomas L. Sobey and Friedrich C. Simmel Abstract Programed molecular structures allow us to research and make use of physical, chemical, and biological effects at the nanoscale. They are an example of the “bottom-up” approach to nanotechnology, with structures forming through self-assembly. DNA is a particularly useful molecule for this purpose, and some of its advantages include parallel (as opposed to serial) assembly, naturally occurring “tools,” such as enzymes and proteins for making modifications and attachments, and structural dependence on base sequence. This allows us to develop one, two, and three dimensional structures that are interesting for their fundamental physical and chemical behavior, and for potential applications such as biosensors, medical diagnostics, molecular electronics, and efficient light-harvesting systems. We describe five tech- niques that allow one to assemble and image such structures: concentration measurement by ultraviolet absorption, titration gel electrophoresis, thermal annealing, fluorescence microscopy, and atomic force microscopy in fluids. Key words: DNA, Self-assembly, Atomic force microscopy, Fluorescence microscopy, Nanostructures Many of the properties that make DNA useful for genetic infor- mation transfer also make it useful for self-assembly of nanostruc- tures. Researchers from physics, chemistry, biology, and computer science use DNA self-assembly to examine the fundamental theo- ries and optimal conditions of self-assembly (1), cooperative effects and emergence; thermodynamics and mechanics of poly- mers (2–4); and biochemical algorithm execution, logic modules and circuits, error correction techniques, and computational demonstrations (5, 6). DNA as a molecule has several advantages when compared with other molecules. It is simple enough to be relatively well 1. Introduction
- 31. 14 Sobey and Simmel understood (compared with proteins), complex enough to build technically advanced structures (compared with many natural and artificial polymers); it can be chemically synthesized (and nowa- days ordered from companies over the internet); and it is stable, reliable, and predictable enough to be confidently handled by researchers with little in the way of chemistry background. A major challenge in the field to date has been coping with exact stoichiometry requirements needed for high numbers of and/or physically large perfectly assembled structures. Typically, several different short (~5–100 bases) strands bind to each other, and if they are not at the correct absolute and relative concentrations then significant defects occur in the final assem- bled structures. Three ideas have been introduced in recent years to overcome this problem. Structures have been assembled that require only one carefully designed sequence which takes advantage of sequence symmetry principles (7). Examples of this include single-sequence DNA nanotubes shown in Figs. 3 and 4. Two other techniques: error avoidance protocols and DNA “origami” that have been also introduced are left for another discussion (8–10). To face the challenges of stoichiometry, one measures the absolute or relative concentrations of DNA very precisely. This is done in one of the two ways. The absorption of DNA at a light wavelength of 260 nm is dependent on its concentration, base sequence length and structure. If the DNA has a known base sequence and length, and does not have any structure (secondary structure), then its concentration can be related to its absorp- tion (11). If the DNA does have some structure, then the correct con- centration ratio with its complementary strands can be chosen by mixing it at different ratios (“titrating”), and analyzing these using titration gel electrophoresis (12). Having determined the concentrations of the DNA strands, they can then be mixed in appropriate buffer conditions and slowly annealed over several days from ~90 to 20°C to assemble the desired structures. To visualize the structures several options may be used (see Note 1). Fluorescent molecules that bind to DNA may be added and the structures viewed with a fluorescence microscope, which is relatively quick and easy. Significantly more challenging is to use an atomic force microscope and fluid cell, visualizing the structures using a scanning probe. This provides much higher resolution. Several examples of structures that are relatively stable and/ or simple to assemble have been developed by Mao and col- leagues. These include single sequence lattices and nanotubes and structures consisting of three sequences, including lattices and polyhedra (7, 13–15).
- 32. 15 Protocols for Self-Assembly and Imaging of DNA Nanostructures The following lists of materials and equipment are suggested, along with recommended suppliers. There are often many other good suppliers for these, the following are suggestions only, in particular with regards to equipment. All water used should be 18 MW and of pH 7–8. 1. UV spectrometer (see Notes 2 and 3) (we use a V-630Bio, Jasco, Japan). 2. Cuvettes: 2 (Hellma, Germany). 3. Ultrapure water: 18 MW, pH 7–8. 4. Pipettes: 100, 2.5 mL (Eppendorf, Germany). 5. Centrifuge tubes: 0.5 mL (Eppendorf, Germany). 6. Clean compressed air/nitrogen and/or lens cleaning tissue. 7. DNA (Integrated DNA Technologies, USA). 1. Gel electrophoresis system (such as the PerfectBlue Dual Gel System, Peqlab Biotechnologie, Germany). 2. Electrophoresis Power supply (such as the EPS301, GE Healthcare, USA). 3. Circulating cooling water at 4°C (see Note 4). 4. Detergent (such as 1104-1, Alconox, USA). 5. Ethanol in squirt dispenser. 6. Acrylamide–bisacrylamide: (Rotiphorese Gel 40, Roth, Germany). Warning: Acryilamide is a neurotoxin and carcin- ogen and should be handled with care in a fume cupboard. 7. 10× TAE Buffer: 400 mM Tris–acetate, 10 mM EDTA, pH 8.3. 8. Ultrapure water: 18 MW, pH 7–8. 9. MgCl2 : 1 M (Sigma-Aldrich, Germany). 10. TEMED (Tetramethylethylenediamine, Sigma-Aldrich, Germany). 11. APS: (Ammonium persulfate, Sigma-Aldrich, Germany) pre- pare fresh solutions weekly at 10% w/v in water. 12. Glass beakers: 2, 150 mL (Duran Group, Germany). 13. Pipettes: 100, 2.5 mL (Eppendorf, Germany). 14. Vacuum chamber and pump (such as model 2478257, Duran Group, Germany, or model MVP 015-4, Pfeiffer Vacuum, USA). 15. Aspirating pipettes: 25 mL (BD Falcon, USA). 2. Materials 2.1. Absolute Concentration by Ultraviolet Absorption Measurements 2.2. Relative Concentration by Polyacrylamide Gel Electrophoresis
- 33. 16 Sobey and Simmel 16. Pipette Filler (VWR International, USA). 17. Bulldog clips. 18. Centrifuge tubes: 0.5 mL (Eppendorf, Germany). 19. 20 mL Syringe. 20. Needle (G 14 0.60×30 mm). 21. Gel loading buffer: 4 g sucrose, 25 mg bromophenol blue, 25 mg xylene cyanol, 25 mg Orange G (Sigma-Aldrich, Germany), H2 O to 10 mL. Store in small aliquots at 4°C. 22. DNA ladder (Low Molecular Weight, such as N3233, New England Biolabs, USA). 23. SYBR Gold (Invitrogen, USA). This is toxic and should be han- dled carefully according to the manufacturer’s instructions. 24. Stiff plastic/card sheet larger than the gel plates. 25. Aluminum foil. 26. Staining tray: opaque plastic box with lid slightly larger than the size of the gel. 27. Gel documentation system (Molecular Imager Gel Doc XR, Bio-Rad, USA). 28. DNA strands (Integrated DNA Technologies, USA). 1. 10× TAE buffer: 400 mM Tris–acetate, 10 mM EDTA, pH 8.3. 2. MgCl2 : 1 M (Sigma-Aldrich, Germany) (see Notes 5 and 6). 3. Water: 18 MW, pH 7–8. 4. Membrane filter: 0.02 mm (Anotop 25 Plus, Whatman, England). 5. Beaker: 2–4 L (Duran Group, Germany). 6. Styrofoam box to fit beaker (see Note 7). 7. Boiling water to fill beaker (see Note 8). 8. Screw-top microtubes: 0.5 mL (VWR International, USA). 9. Zip-lock bag. 10. Metal weights (nuts and bolts). 11. Glass thermometer 0–100°C. 12. Pipettes: 100, 2.5 mL (Eppendorf, Germany). 13. DNA strands (Integrated DNA Technologies, USA). 1. Fluorescence microscope (see Note 9) (Olympus IX71, Olympus, Japan). 2. 10× TAE buffer: 400 mM Tris–acetate, 10 mM EDTA, pH 8.3. 3. Water: 18 MW, pH 7–8. 2.3. Thermal Annealing of DNA Nanostructures 2.4. Fluorescence Microscopy of DNA Nanostructures
- 34. 17 Protocols for Self-Assembly and Imaging of DNA Nanostructures 4. YOYO-1 (Invitrogen, USA). Warning: This is toxic and should be handled carefully according to the manufacturer’s instructions. 5. Microscope slides or cover slips, thickness 0 (Menzel, Germany). 6. Fingernail varnish. 7. Wavelength filter (U-MWIB2, Olympus, Japan). 8. Light source (X-Cite Series 120, EXFO Photonic Solutions, Canada). 9. Ascorbic acid (see Note 10). 10. Pipettes: 100, 2.5 mL. 11. DNA product. 1. Atomic force microscope (we use a Multimode V, Veeco Instruments, USA): operated in intermittent contact (tapping) mode. 2. Fluid cell (Veeco Probes, USA). 3. Mica (50, Ted Pella, USA). 4. Metal puck (Ted Pella, USA). 5. Cantilevers (model DNP-S10, Veeco Probes, USA). 6. 10× TAE buffer: 400 mM Tris–acetate, 10 mM EDTA, pH 8.3. 7. Ultrapure water: 18 MW, pH 7–8. 8. Membrane filter: 0.02 mm (Anotop 25 Plus, Whatman, England). 9. Optical microscope. 10. Tweezers (such as model 5599, Ted Pella, USA). 11. Pipettes: 100, 2.5 mL (Eppendorf, Germany). 12. DNA product. 1. Single-stranded DNA can have significant secondary struc- ture (where bases in the same strand bind to each other). This alters the extinction coefficient and leads to incorrect concen- tration determination. With current models and technology, there is no way around this (apart from using strands that are designed not to have secondary structure) and the best way to circumvent this is to use titration gel electrophoresis. However, titration gel electrophoresis requires much more time and effort, and thus is usually only conducted when it is found the 2.5. Atomic Force Microscopy in Fluid of DNA Nanostructures 3. Methods 3.1. Absolute Concentration by Ultraviolet Absorption Measurements
- 35. 18 Sobey and Simmel lattice is not forming as desired from the concentrations determined by UV absorption measurements. 2. Turn on spectrometer; allow lamp and system to stabilize for 1–2 h. 3. Appropriate DNA sequences can be dissolved in water to a concentration of 100 mM, this can be determined from the information sheet accompanying the sequences. These should be briefly heated to 60°C and well vortexed to ensure complete mixing. 4. Calculate a molar extinction coefficient for each DNA sequence using the nearest-neighbor model (16–18) – for example using Scitools on the internet from Integrated DNA Technologies (see Note 11). 5. Rinse the cuvette under flowing water, shake water out by hand hard, repeat several times. Dry the outer surface with compressed air/nitrogen and lens cleaning tissue. 6. Load the cuvettes with 100 mL water, set parameters (depend- ing on the model of spectrometer, these exact options may not be possible, but there should be similar possibilities): (a) Wavelength scan: 350–220 nm (b) Scan rate: 400 nm/min (c) Bandpass: 1 nm (d) Response (integration) time: medium 7. Measure baseline, set baseline subtraction. 8. Add 2 mL of DNA to the measurement cuvette without removing it from the spectrometer, stir with pipette tip for 10–20 s. 9. Measure absorbance, ensure that the absorbance lies between 0.1 and 1 or add or dilute DNA until this is the case. Also ensure that absorbance between 320 and 350 nm is extremely close to zero or apply an offset if it is not, read-off absorbance at 260 nm (note this may not be the peak maximum), (see Notes 12 and 13) see Fig. 1 for an example. 10. Calculate the concentration of DNA using the Beer-Lambert law: Absorbance path length extinction coefficient concentration = ´ ´ (1) absorbance Concentration path length extinction coefficient = ´ (2) For example, with an absorbance of 0.5 and an extinction coef- ficient of 100,000 L/mol·cm and a cuvette of width 1 cm:
- 36. 19 Protocols for Self-Assembly and Imaging of DNA Nanostructures Fig.1. A representative ultraviolet absorbance curve of a single-stranded DNA sequence. Note that the maximum is not exactly at a wavelength of 260 nm (this is dependent on the sequence); however, the absorbance is measured at 260 nm because this is the value that the extinction coefficient is normally calculated at. Note also that a baseline is measured directly before adding DNA, thus there is no vertical offset necessary, and this can be seen by the 0 values at higher wavelengths (320–350 nm). 0.5 Concentration 5 M 1cm 100,000(L/mol cm) = = m ´ (3) 11. Calculate the amount of water needed to be added achieve a concentration of 1 mM (the DNA nanostructures are generally assembled at a DNA concentration of 1 mM or less). Continuing the example: initial initial final final Concentration volume concentration volume ´ = ´ (4) initial initial final final concentration volume Volume concentration ´ = (5) final 5 M 102 L Volume 510 L 1 M m ´ m = = m m (6) needed Volume 510 102 L 408 L. = - m = m (7) Add this amount and mix with pipette tip. 12. Transfer solution to a centrifuge tube (loss of small amounts here is not critical, if well mixed, the concentration will not change). 13. Repeat steps from three onward for all DNA strands.
- 37. 20 Sobey and Simmel 1. Clean electrophoresis plates thoroughly with detergent and rinse thoroughly with water, wipe with ethanol then wipe dry. 2. Place plates with spacers together and set in the electrophoresis unit, with the gap for the comb upward and inward. 3. Squirt ethanol in between the plates until approximately 1/4 full and leave for several minutes to ensure there are no leaks. 4. Mix Acrylamide–bisacrylamide, TAE buffer, MgCl2 solution and water in the following ratio per 10 mL of resulting solution (see Note 14) (Table 1). 5. Place in vacuum chamber for 5 min to remove air from the solution (this speeds up polymerization). 6. Check electrophoresis plates to see that there are no leaks, pour out ethanol. If there are leaks, pull the plates apart, put them back together again and recheck. 7. Remove solution from vacuum chamber. Divide solution gen- tly into two (for two gels) without mixing in unnecessary air. 8. Prepare pipettes and tips for the APS and TEMED solutions. 9. Under the fume-hood, working without pause, add APS solution at 50 mL per 10 mL of solution to the first flask, and swirl gently to mix. Add TEMED (closing TEMED lid imme- diately) at 10 mL per 10 mL of solution to the second flask, and swirl gently to mix. 10. Immediately pipette (slowly to avoid bubbles) solution between the first set of gel electrophoresis plates, making sure no air bubbles get trapped. Fill until the level reaches the bot- tom of the gap for the comb. 11. Insert the comb, ensure that it traps no air bubbles, if this is the case take it out and reinsert it. It is useful to have the top of the comb slightly (~1 mm) above the upper edge of the glass plates (see Note 15). Use bulldog clips to hold the comb securely into position (otherwise, the expanding polymeriz- ing gel displaces it). 12. Repeat steps 9–11 for the second solution. 13. Optimally, wait 90 min for the gel to polymerize (shorter times and the gel does not have polymerized completely with even and static pore sizes, longer than a couple of hours and the gel swells and dries) (see Note 16). 3.2. Relative Concentration by Polyacrylamide Gel Electrophoresis Table 1 Pipetting instructions for 10 mL of a 20% TAE/Mg2+ polyacrylamide gel Polyacrylamide gel (%) Acrylamide–bisacrylamide (37.5:1) (mL) 10 ¥ TAE (mL) 1 M MgCl2 (mL) H2 O (mL) 20 5.00 1 120 3.88
- 38. 21 Protocols for Self-Assembly and Imaging of DNA Nanostructures 14. Fill buffer between the gels and into the reservoirs of the electrophoresis unit. 15. Remove one of the combs and immediately flush the wells completely with buffer using the syringe and needle to remove unpolymerized acrylamide (see Note 17). 16. Repeat for second comb. 17. Set out nine 0.5 mL centrifuge tubes in a holder. 18. Calculate – from the concentrations (in mM) determined by UV absorption – the volume of the first strand needed for 100 ng (shorter strands run relatively faster and spread rela- tively wider, thus may need to be relatively more concentrated when run with longer strands). Use the formula: 9 100 10 g Volume( L) molecular mass(g/mol) concentration(mol/ L) - ´ m = ´ m (8) Add this volume of the first strand to each tube. 19. Calculate the quantity in moles of the first DNA strand that this volume holds using: Quantity concentration volume = ´ (9) 20. Calculate the volume needed of the second strand for each these (suggested) factors of the first DNA strand: 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2. quantity Volume factor concentration = ´ (10) 21. Add each volume to one of the tubes (there is one tube left). 22. Calculate the amount of 1 M MgCl2 solution needed to be added to each tube to give a concentration of 12.5 mM. Add this amount to each respective tube. 23. Close the tubes, label them, and vortex briefly. 24. Using a Polymerase Chain Reaction (PCR) machine, heat tubes to 90°C (lid temperature 92°C) for 1 min and cool evenly in small steps to room temperature over 1 h. This should ensure hybridization of the strands. 25. Add 0.4 mL of low molecular weight DNA ladder to the remaining tube. Pipette 3 mL of loading buffer into each tube and vortex. 26. Prepare running buffer and salt (1 L or more depending on the size of the electrophoresis unit): 20 mL 50× TAE buffer, 12.5 mL 1 M MgCl2 , and water to fill to 1 L. Refrigerate until at 4°C. The buffer stock should be the same as that used for the gel and at the same concentration (1X). 27. Flush wells again with buffer using the syringe and needle, immediately before loading wells with DNA.
- 39. 22 Sobey and Simmel 28. Load the first well with prepared DNA ladder solution and the rest of the wells with the DNA strand solutions in order. 29. Connect the electrophoresis unit to the circulating cooling water. This ensures that the gels remain cool while running and do not thermally denature the hybridized DNA samples (see Note 4). 30. Connect the electrophoresis unit to the power supply and run at a constant voltage of 10 V per cm gel length until the yellow loading dye runs to the bottom of the gel (typically 1–3 h). 31. During this time, the staining solution can be prepared. An opaque plastic container with a flat bottom just larger than the gel is needed, and this is filled with buffer to a depth that would be the same as the thickness of the gel. SYBR Gold is added at a ratio of 1 mL per 10 mL, this is covered with an opaque lid or aluminum foil and allowed to mix on a rotator at a small angle to the horizontal at the lowest speed (1 Hz). 32. Prepare a sheet of aluminum foil about three times the size of the gel flat on the table. 33. When the gel is finished, turn off the power supply, and remove the gel from the unit. Use a thin blade or plastic scraper to carefully remove the top gel plate. A few droplets of water between the plate and the gel can help. Spread the aluminum foil over the top of the gel, then place a flat stiff piece of plastic over the aluminum foil. Use this to support the gel “sandwich,” as it is flipped up so that the bottom gel plate is now on top. This plate is also removed, two opposite sides of the foil are trimmed to the gel width, the remaining two sides are used to lift and support the gel, and the whole lot is placed in the staining container. The lid is placed on and the gel is left to stain on the rotator for 30–60 min. 34. The rotator is then stopped, the staining solution is removed using the 25 mL pipette and disposed of as toxic waste. 35. The gel is lifted out using the aluminum foil support and onto the UV light box. The gel can then be slid off the aluminum foil onto the glass using a few drops of water as lubricant if necessary. 36. The aluminum foil is stored safely for next time or disposed of as toxic waste. 37. The gel is examined/photographed using the gel documenta- tion system and an appropriate wavelength filter for SYBR Gold making sure the focal distance of the camera is set to reach the gel and not to the inner UV bulbs (see Fig. 2 for an example). 38. The correct ratio of DNA strands is chosen by comparing the bands to see the one-to-one binding ratio. This ratio can be used with the excess (not used in the gel analysis) DNA to self-assemble the desired structure.
- 40. 23 Protocols for Self-Assembly and Imaging of DNA Nanostructures 1. The DNA strands are mixed at the correct concentrations and thermally annealed in appropriate buffer conditions (see Note 18). 2. All stock buffer and salt solutions should be filtered with a 0.02 mm membrane filter before use, this is quite critical. 3. The final volume and concentration of required DNA prod- uct is decided. Typically, volumes between 20 and 2,000 mL are produced, at concentrations between 50 and 1,000 nM. As an example, 100 mL of 5-stranded lattice at 500 nM total concentration is chosen. 4. The concentration needed of each strand is calculated, this depends on the stoichiometry of the strands in the final structure. stoichiometry of strand Concentration of strand total stoichiometry total concentration = ´ (11) 5. For a strand mixed at a ratio of 1 with a total of five strands, this is 1 Concentration of strand 500 nM 100 nM 5 = ´ = (12) 6. The volume needed from each DNA strand solution is calcu- lated using Eq. 4. For example, if each strand solution has been diluted to 1,000 nM, then for 100 mL final volume: initial 1,000nM volume L 100 nM 100 L ´ m = ´ m (13) 3.3. Thermal Annealing of DNA Nanostructures Fig. 2. A native PAGE gel electrophoresis titration analysis of the concentration of two complementary DNA strands. Each strand is 8 bases long, the gel is 20% and was run at 10 V/cm for 3 h. Lane 1: low molecular weight DNA ladder (766–25 bp); Lanes 2–9: relative concentration increments from factors of 2 to 0.25 as listed in the protocol. The upper band represents the hybridized DNA, the lower band excess single-stranded DNA: Lane 6 has the correct ratio of the two strands in this case, as there is no excess single- stranded DNA.
- 41. 24 Sobey and Simmel initial 100 nM 100 L Volume 10 L 1,000 nM ´ m = = m (14) 7. The calculated volume of each strand is pipetted into a screw- top microcentrifuge tube. 8. The volume of 50× TAE buffer needed for a 1× solution of 100 mL is calculated using the Eq. 4. For example: initial 50 volume L 1 100 L. ´ m = ´ m (15) initial 1 100 L Volume 2 L 50 ´ m = = m (16) This volume is pipetted into the screw-top microcentrifuge tube. 9. The volume of 1,000 mM (1 M) MgCl2 solution needed for a 12.5 mM solution of 100 mL is initial 1,000 mM volume L 12.5 mM 100 L. ´ m = ´ m (17) initial 12.5 mM 100 L Volume L 1.25 L 1,000 mM ´ m = m = m (18) This is pipetted into the screw-top microcentrifuge tube. 10. Enough water is added (here 46.75 mL) to make up the total required volume (here 100 mL). 11. The lid is screwed tightly onto the tube, and it is briefly (10 s each) centrifuged, vortexed, then centrifuged again. 12. The tube is placed into the zip-lock plastic bag with enough weights (nuts and bolts) to make sure it sinks, the bag is rolled up and secured with a rubber band, and a few small holes are made in it to allow the air to escape. 13. Enough (tap) water is boiled to fill the large (2–4 L) beaker. The bag with weights and microcentrifuge tube is placed in the bottom of the beaker, along with the thermometer. The beaker is filled with water just below boiling point. 14. The beaker is placed inside the Styrofoam box which is closed, and this is placed in a safe place and left for 48 h or until the water has cooled to room temperature (~20°C). 15. The microcentrifuge tube is then taken out and dried, to ensure that no water droplets on the outside enter upon opening the lid of the microcentrifuge tube. 1. If the chosen DNA structure has dimensions on the order of several micrometers or greater (for example, a large two- dimensional lattice), then it may be viewed with a fluores- cence microscope if it is “dyed” using an intercalating 3.4. Fluorescence Microscopy of DNA Nanostructures
- 42. 25 Protocols for Self-Assembly and Imaging of DNA Nanostructures fluorescent molecule, such as YOYO-1 (Invitrogen). This binds between base pairs of double-stranded DNA, optimally at a ratio of 1 dye molecule per 5 base pairs (see Note 19). 2. Thus, if the total number of bases of the 5 strands is 100, then the number of base pairs is 50. The YOYO-1 stock solution is 1,000,000 nM (1 mM) and typically one dyes a DNA struc- ture solution of 10 mL. The volume of YOYO-1 needed is 50 base pairs 1,000,000 nM volume L initial 5 500 nM 10 L ´ m = ´ ´ m (19) initial (50 base pairs/5) 500 nM 10 L Volume L 1,000,000 nM 0.05 L ´ ´ m = m = m (20) 3. This volume is not realistic to pipette, so the YOYO-1 is diluted in 1× TAE buffer, for example (100× dilution): 0.5 mL YOYO-1 stock solution, 2 mL 50× TAE buffer, 97.5 mL water in a plastic (YOYO-1 binds to glass containers) microcentri- fuge tube. 4. With 100× dilution, 5 mL of this is pipetted into a microcen- trifuge tube. 5. Using a cut-off tip 10 mL of DNA structure solution is added (see Note 20). 6. Ascorbic acid is used to minimize photobleaching of the fluorescent molecules. It is prepared at 100 mM in a volume of, for example, 10 mL. With a molecular mass of 176.12 g/mol this is: Mass molecular mass concentration volume = ´ ´ (21) 3 3 Mass 176.12(g/mol) 100 10 (mol/L) 10 10 L 176 mg - - = ´ ´ ´ ´ = (22) This mass is dissolved in 10 mL of water and stored in a light proof jar. New solutions should be made every week. 7. This is added to a final concentration of 10 mM, so with 5 mL of YOYO-1 solution and 10 mL of DNA solution, one adds approximately 1.5 mL. 8. The fluorescence microscope is prepared, the light source is switched on, the correct filter is loaded, and an appropriate objective (40× air) is chosen. 9. For a very quick look, 1 mL of dyed-DNA solutions can be pipetted using a cut-off tip onto a Number 0 cover slip and placed on the microscope. There are large amounts of back- ground fluorescence, but normally the structures themselves can also be seen.
- 43. 26 Sobey and Simmel 10. For a better image, this process is repeated but the droplet is covered with a second cover slip and the edges of the cover slip are sealed with fingernail varnish. There is much less fluorescence background using this technique. 1. The DNA structures bind in solution to a mica surface given the correct conditions, and the topography of the structure can then be measured/“visualized” using an atomic force microscope. The precise details of this protocol vary greatly depending on the model of atomic force microscope used. 2. The microscope and control computer are switched on, and the software loaded. 3. It is generally much easier to set the correct engage height of the cantilever above the mica surface in air, as the surface of the mica is difficult to see with an optical microscope when submerged in buffer. 4. The cantilever holder is set to a distance far enough from the surface to ensure that upon loading the cantilever the tip of the cantilever does not contact the surface. 5. Using a small optical microscope and tweezers, the tip is loaded correctly in the fluid cell. 6. The mica is loaded into the microscope (initially without any sample). 7. The fluid cell/holder is loaded into the microscope. 8. With the aid of the optical microscope that comes with the atomic force microscope, the laser spot is aligned onto the very end and center of the cantilever. This is important! 9. If there is a reflecting mirror, its angle is adjusted so the laser shines close to the center of the photodiode window. 10. The photodiode position is adjusted so that the laser is reflected directly at its center. 11. The steps 8–11 are repeated to fine-tune the system to ensure that the detected signal is high (with the laser reflecting very close to the end of the tip) and the deflection signal (relating to the laser reflecting onto the center of the photodiode) is minimal. 12. The surface of the mica is brought into focus of the optical microscope. The surface can be difficult to observe (being semitransparent), it can help to move around looking for cracks on the surface. The cantilever is brought to a level just before it comes into perfect focus, indicating that it is very close to, but not in contact with the surface (see Note 21). As a guide, as the cantilever moves closer to the focal height, a double image of the cantilever is seen, and this merges into one at the focal height. 3.5. Atomic Force Microscopy on Fluid of DNA Nanostructures
- 44. 27 Protocols for Self-Assembly and Imaging of DNA Nanostructures 13. The mica is then removed (this probably entails removing the fluid cell/holder also) and a fresh surface is prepared using (opaque) masking tape (see Note 22). This is best done by pressing firmly a strip of tape flat onto the mica on a table, lifting the far edge of the mica up so that it stands perpen- dicular to the table on its bottom edge, and peeling the tape slowly and evenly downward. A thin, complete, shiny layer of mica should have adhered to the tape. Quality of results may depend on the orientation of the tape relative to the mica, there is an optimal direction found by experimenting. 14. 5 mL of DNA structure solution is carefully pipetted onto the center of the mica using a cut-off pipette. 15. 1×TAE12.5mMMgCl2 (filteredthrougha0.02mm membrane filter before use) buffer solution is added to the mica, and/or the surface of the fluid cell/holder and/or through a tube into the fluid cell, dependent on the system. Care should be taken that no air bubbles are trapped on the cantilever. 16. The mica is carefully reloaded into the microscope. 17. The buffer has a different refractive index so that the laser beam travels a slightly different path, steps 8–11 may need to be repeated with small changes to optimize the measured laser signal. 18. The cantilever is tuned (generally using a function in the software) to ~5% below its resonant vibration frequency (see Notes 23 and 24). The amplitudes used are much smaller than those in air, and should be adjusted to be above the level at which the tip sticks to the surface when imaging, but not so large that the sample is damaged by the tip’s vibrations. This is best determined through trial and error. 19. The amplitude set point (ratio of the free amplitude of vibra- tion to the amplitude while imaging) is generally set just below 1, for example 0.98 (or 98%). However, this can vary greatly dependent on the system. 20. The imaging parameters are then set. Initial scan sizes and speeds are set small (1 mM) and slow (0.5 Hz) to prevent damage to the tip as it first “contacts” the surface. 21. The most important two other parameters are the integral and proportional gains, these should be initially set extremely small (exact values are system dependent). 22. The number of measurements per scan line (pixels) can be set to 256. 23. The “engage surface” function of the microscope is actuated. Several errors may occur during this process (see Note 25). 24. When correctly engaged on the surface, the imaging param- eters are optimized. It is generally helpful to first withdraw
- 45. 28 Sobey and Simmel the cantilever from the surface slightly (several hundred nanometers), retune the cantilever to the correct frequency and drive amplitude, before reengaging the surface. 25. There are generally at least two “views” in the software, an “image” view and an “oscilloscope” view of the trace and retrace of the current line scan profile. The amplitude set point, which is slowly increased until the tip just no longer contacts the surface, is the best seen in the oscilloscope mode when the trace and retrace scan profiles significantly depart vertically from each other. It is then decreased to just below the value when they come back vertically on top of each other for optimal imaging. 26. The integral gain is gradually increased so that the trace and retrace scan profiles correlate optimally with one another without excessive noise being introduced into the signals. 27. The proportional gain is then adjusted similarly. 28. The scan size and speed can then be increased and suitable DNA structures for imaging are found. 29. The desired scan size is set, and the scan speed is slowed to 1–3 Hz, and the number of measurements per line is increased to 512 or 1024. An image is then captured. 30. Care should be taken that no imaging artifacts like double tip images (coming from broken tips with two or more points) or material sticking to the tip occur, if so the cantilever should be changed and the whole process repeated. 31. When a new sample is required, the process can be simplified if care is taken. The tip is withdrawn approximately 100 mm from the surface. If the cantilever is not moved within its holder, a thin layer of mica is removed from the same mica sample, and the mica is returned afterward to the same position in the microscope, then the cantilever should be relatively close to the surface and should not need long for the engage procedure. 32. Examples of atomic force microscopy images taken using this method are shown in Figs. 3 and 4. 1. There are other options, such as Transmission Electron Microscopy, that are not discussed here. 2. There are now ultraviolet absorption spectrometer systems that are designed to quickly measure mL volumes in the mM range. These may not be accurate enough for the standards required here. 4. Notes
- 46. 29 Protocols for Self-Assembly and Imaging of DNA Nanostructures 3. For DNA concentration measurements, temperature control (via a programmable water bath or Peltier element) of the sample while measuring absorption is not usually necessary; however, this is useful for making DNA melting measure- ments, often used to assist in analyzing these structures. 4. The gels should be “run” at 4°C, and this can also be achieved by placing the system in a cool room or refrigerator. Fig. 3. An atomic force microscopy image of self-assembled DNA nanotubes that have clumped together. Excess DNA that did not form nanotubes can be seen as a back- ground carpet.This height image was captured using “tapping mode” in buffer on mica. Scale bar 1 mm, height scale 15 nm. Fig. 4. An atomic force microscopy image of a self-assembled DNA nanotube that has connected both its ends (by chance). Thinner-tangled nanotubes can also be seen. This height image was captured using “tapping mode” in buffer on mica. Scale bar 500 nm, height scale 30 nm.
- 47. 30 Sobey and Simmel 5. Salts are critical for these structures – they provide electrostatic shielding that allows the negative DNA strands to bind together. In standard DNA hybridization, salts with monova- lent ions like Sodium Chloride are used. For the structures discussed here, salts with divalent ions such as MgCl2 are used, and these allow the DNA to “fold” into the desired complex structures. 12.5 mM concentration is generally chosen, and this is high enough for binding and folding and allows the large DNA structures to bind to the mica surface in atomic force microscopy. Higher volumes may cause condensation of the DNA or unwanted significant binding of any excess single-stranded DNA to the mica surface. 6. Alternatively, a large thermos flask can be used instead of a beaker and polystyrene box. 7. Instead of annealing in hot water, a programmable PCR machine can be used with small temperature steps, ensuring that the lid is a few degrees warmer than the heating block. 8. This can be normal tap water. 9. A Total Internal Reflection Fluorescence (TIRF) microscope is advantageous to remove background fluorescent light from sources not in focus (at the surface), but imaging is certainly manageable without such a system. 10. This helps to prevent photobleaching of the fluorescent molecules. 11. The extinction coefficient is calculated using a nearest- neighbor model. One can, for example, make use of the online calcula- tor “Scitools” provided by Integrated DNA Technologies at http:// www.idtdna.com/analyzer/Applications/ OligoAnalyzer/ 12. The absorption spectrum of DNA is sequence dependent, and thus the UV absorption peak of DNA may be found between approximately 260±15 nm; however, the absorption should be measured at the wavelength that the extinction coefficient is calculated for, which is generally 260 nm. 13. Depending on the cuvette, it may be necessary to stir the sample with a pipette tip to remove an air bubbles and make a second measurement to ensure reproducibility. 14. This gel concentration is suitable for DNA strands up to 100 bases long, for longer strands smaller gel concentrations are needed. 15. The gels can be stored for several days in their glass plates if wrapped with tensioned rubber bands to keep the comb pressed securely into the wells (with the upper edge of the comb above the glass taking some of the tension) and kept in buffer. Metal clips oxidize in buffer and should not be used.
- 48. 31 Protocols for Self-Assembly and Imaging of DNA Nanostructures 16. Gels may be stored for several days if wrapped securely with rubber bands – ensure that there is tension holding the comb correctly in place; otherwise, the wells fill with unpolymer- ized acrylamide – and stored in 1× TAE solution. 17. A small battery head lamp can help to make the gel wells more visible (Petzl Tikka). 18. We have also developed a technique that does not rely on thermal annealing but rather on the basis of dilution of DNA denaturing agents in the buffer (19). 19. One YOYO-1 molecule every five base pairs gives the best ratio of minimal structural deformation of the DNA helix to maximal fluorescence intensity, giving an optimal signal to noise ratio (20). 20. The DNA structures can be so large that the normal hole diam- eter of the pipette tip damages them as they pass through. 21. It is important to come into focus on the surface from a starting point far away from the surface; otherwise, one may focus on the reflection and not on the real surface. 22. One can see the thin peeled layers of mica with better contrast if the tape is opaque. 23. The feedback loop in the electronics of the microscope works optimally at values just below the resonance frequency of the cantilever. 24. In the 10 kHz range with small buffer volumes, there may be a resonance in the buffer itself which can be heard as a high-pitched tone. This is normal. 25. Several errors often occur while engaging, if these occur, the engage should be aborted. The amplitude may change significantly (more than 10%), thus the cantilever should be retuned with the correct amplitude. The deflection errors may increase significantly, particularly if the buffer was ini- tially at a different temperature to the fluid cell/holder and/ or mica, thus the errors should be brought to a minimum. Once corrected, the engage can be restarted. Acknowledgments The authors sincerely thank Helene Budjarek for her technical expertise and assistance, and Rob Fee and Ralf Jungmann for helpful discussions. The authors acknowledge financial support from the Center for Nanoscience (Ludwig-Maximilian- Universität, Germany), the International Doctorate Program NanoBioTechnology (Elite Network of Bavaria), and the Nanosystems Initiative Munich.
- 49. 32 Sobey and Simmel References 1. Pelesko, J. A. (2007) Self Assembly: The Science of Things That Put Themselves Together, Chapman Hall/CRC. 2. Zheng, J. W., Lukeman, P. S., Sherman, W. B., Micheel, C., Alivisatos, A. P., Constantinou, P. E., and Seeman, N. C. (2008) Metallic Nanoparticles Used to Estimate the Structural Integrity of DNA Motifs, Biophys. J. 95, 3340–3348. 3. Green, S. J., Bath, J., and Turberfield, A. J. (2008) Coordinated Chemomechanical Cycles: A Mechanism for Autonomous Molecular Motion, Phys. Rev. Lett. 101. 4. Dirks, R. M., Bois, J. S., Schaeffer, J. M., Winfree, E., and Pierce, N. A. (2007) ThermodynamicAnalysisofInteractingNucleic Acid Strands, SIAM Review 49, 65–88. 5. Seelig, G., Soloveichik, D., Zhang, D. Y., and Winfree, E. (2006) Enzyme-Free Nucleic Acid Logic Circuits, Science 314, 1585–1588. 6. Zhang, D. Y., Turberfield, A. J., Yurke, B., and Winfree, E. (2007) Engineering Entropy- Driven Reactions and Networks Catalyzed by DNA, Science 318, 1121–1125. 7. Liu, H. P., Chen, Y., He, Y., Ribbe, A. E., and Mao, C. D. (2006) Approaching the Limit: Can One DNA Oligonucleotide Assemble into Large Nanostructures?, Angew. Chem.- Int. Edit. 45, 1942–1945. 8. Soloveichik, D., Cook, M., and Winfree, E. (2008) Combining Self-Healing and Proofreading in Self-Assembly, Natural Computing 7, 203–218. 9. Shih, W. M., Quispe, J. D., and Joyce, G. F. (2004) A 1.7-Kilobase Single-Stranded DNA That Folds into a Nanoscale Octahedron, Nature 427, 618–621. 10. Rothemund, P. W. K. (2006) Folding DNA to Create Nanoscale Shapes and Patterns, Nature 440, 297–302. 11. Tataurov, A. V., You, Y., and Owczarzy, R. (2008) Predicting Ultraviolet Spectrum of Single Stranded and Double Stranded Deoxyribonucleic Acids, Biophys. Chem. 133, 66–70. 12. Lu, M., Guo, Q., Marky, L. A., Seeman, N. C., andKallenbach,N.R.(1992)Thermodynamics of DNA Branching, J. Mol. Biol. 223, 781–789. 13. Zhang, C., He, Y., Chen, Y., Ribbe, A. E., and Mao, C. D. (2007) Aligning One-Dimensional DNA Duplexes into Two-Dimensional Crystals, J. Am. Chem. Soc. 129, 14134-+. 14. He, Y., Chen, Y., Liu, H. P., Ribbe, A. E., and Mao, C. D. (2005) Self-Assembly of Hexagonal DNA Two-Dimensional (2d) Arrays, J. Am. Chem. Soc. 127, 12202–12203. 15. He, Y., Ye, T., Su, M., Zhang, C., Ribbe, A. E., Jiang, W., and Mao, C. D. (2008) Hierarchical Self-Assembly of DNA into Symmetric Supramolecular Polyhedra, Nature 452, 198–U141. 16. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Predicting DNA Duplex Stability from the Base Sequence, Proc. Natl. Acad. Sci. U. S. A. 83, 3746–3750. 17. Sugimoto, N., Nakano, S., Yoneyama, M., and Honda, K. (1996) Improved Thermodynamic Parameters and Helix Initiation Factor to Predict Stability of DNA Duplexes, Nucleic Acids Research 24, 4501–4505. 18. SantaLucia, J., Allawi, H. T., and Seneviratne, A. (1996) Improved Nearest-Neighbor Parameters for Predicting DNA Duplex Stability, Biochemistry 35, 3555–3562. 19. Jungmann, R., Liedl, T., Sobey, T. L., Shih, W., and Simmel, F. C. (2008) Isothermal Assembly of DNA Origami Structures Using Denaturing Agents, J. Am. Chem. Soc. 130, 10062–10063. 20. Doyle, P. S., Ladoux, B., and Viovy, J. L. (2000) Dynamics of a Tethered Polymer in Shear Flow, Phys. Rev. Lett. 84, 4769–4772.
- 50. 33 Giampaolo Zuccheri and Bruno Samorì (eds.), DNA Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 749, DOI 10.1007/978-1-61779-142-0_3, © Springer Science+Business Media, LLC 2011 Chapter 3 Self-Assembly of Metal-DNA Triangles and DNA Nanotubes with Synthetic Junctions Hua Yang, Pik Kwan Lo, Christopher K. McLaughlin, Graham D. Hamblin, Faisal A. Aldaye, and Hanadi F. Sleiman Abstract The site-specific insertion of organic and inorganic molecules into DNA nanostructures can provide unique structural and functional capabilities. We have demonstrated the inclusion of two types of mole- cules. The first is a diphenylphenanthroline (dpp, 1) molecule that is site specifically inserted into DNA strands and which can be used as a template to create metal-coordinating pockets. These building blocks can then be used to assemble metal-DNA 2D and 3D structures, including metal-DNA triangles, described here. The second insertion is a triaryl molecule that provides geometric control in the prepara- tion of 2D single-stranded DNA templates. These can be designed to further assemble into geometrically well-defined nanotubes. Here, we detail the steps involved in the construction of metal-DNA triangles and DNA nanotubes using these methods. Key words: DNA, Self-assembly, Nanostructure, Transition metal, Nanotube The field of structural DNA nanotechnology has evolved a number of elegant strategies for organizing materials on the nanometer length scale (1–3). By taking advantage of the information-rich biomolecule DNA, simple self-assembly can now be used to prepare unique structures in multiple dimensions and template the arrangement of more functional components with remarkable precision and control. While DNA represents one of nature’s most predictable self- assembling systems, greater structural and functional diversity can be realized through strategic modification using small organic and 1. Introduction
- 51. 34 Yang et al. inorganic molecules. Such molecules can augment the non-covalent interactions of nucleic acids and provide chemical advantages in the form of enhanced stabilization, and photophysical, redox, magnetic, and catalytic properties that are not observed naturally for DNA. Conceptually, this brings the toolbox of supramolecular chemistry and the ability of this field to generate a diverse array of structures and functions using synthetic molecules, and combines this area with the programmability of DNA. We recently introduced the term “supramolecular DNA assembly” to describe the melding of these two fields, whereby the self-assembly properties of DNA are com- plemented by those of synthetic components that have been site specifically inserted within the nucleic acid components (2). This approach is in contrast with more conventional DNA assembly methods that rely entirely on unmodified DNA, and may allow for unique structural and functional diversity. In this chapter, we describe the necessary steps to build two DNA nanostructures using such small synthetic molecule- modified DNA. In one case, a DNA triangle acts as a template for site-specific metal incorporation (4), while in the other, it becomes a building block for the assembly of geometrically well-defined nanotubes (5). Organic molecules are first made compatible with solid-phase DNA synthesis via conversion to phosphoramidite derivatives, and incorporated as vertices or ligands within oligonucleotides that are designed to form discrete polygons (4, 6). The assembly process yields a single-stranded template for either immediate functionalization with transition metals or as a module used as the starting point for nanotube preparation. Such methodology utilizes the properties of both DNA and synthetic molecules in tandem to prepare dynamic nanoscale two-dimensional (2D) and 3D products that are formed quantitatively. The first system com- bines the programmability of DNA with the redox, photoactivity, and magnetic properties of transition metal complexes. The sec- ond system allows the creation of DNA nanotubes of deliberately controlled geometries, for application as selective host structures, as templates for nanowire fabrication, and as drug delivery tools. 1. Standard reagents and phosphoramidites for automated solid- phase DNA synthesis (see Note 1). 2. 3′-Phosphate functionalized controlled pore glass (CPG) (Chemgenes) with 1,000 angstrom pore size and loading densities of ca. 30 mmol/g. 2. Materials 2.1. Solid-Phase DNA Synthesis and Purification
- 52. 35 Self-Assembly of Metal-DNA Triangles and DNA Nanotubes with Synthetic Junctions 3. Phosphoramidite derivatives of 1 and 2 are prepared as previously reported (see Note 2). 4. 5-Ethylthio-1H-tetrazole (ETT, Sigma–Aldrich). 5. Acetonitrile - low water (EMD). 6. Concentrated ammonium hydroxide (NH4 OH) solution (28%, Fisher Scientific). 7. Sephadex G-25 (superfine DNA grade) (Amersham Bio sciences) in pre-packed columns. 1. TB (10×): 900 mM tris (hydroxymethyl)-aminomethane (Tris) and 900 mM boric acid, pH 8. Store at room tempe rature. 2. TB (1×): prepared by tenfold dilution of TB (10×). 3. TAMg (10×): 400 mM Tris, 76 mM MgCl2 ×6H2 O, and 14 mM glacial acetic acid, pH 8 (adjusted with small amounts of glacial acetic acid). 4. TAMg (1×): prepared by tenfold dilution of TAMg (10×). 5. 40% Acrylamide/bis solution (Fisher Scientific) (WARNING: the unpolymerized solution is a neurotoxin and care should be taken to avoid exposure) and N,N,N′,N′- tetramethylethylenediamine (TEMED, Sigma–Aldrich). 6. Denaturing polyacrylamide gel electrophoresis (PAGE) solution (24%, 100 mL): mix 42.04 g urea, 10 mL TB (10×), and 60 mL 40% acrylamide solution and add water to adjust the volume to 100 mL. 7. Native PAGE solution (8%, 100 mL): mix 10 mL TAMg (10×) and 20 mL 40% acrylamide solution and add water to adjust the volume to 100 mL. 8. Ammonium persulfate (APS). 9. Gel combs: preparative 1.5-mm-thick single lane comb (Hoefer) and 15-lane 0.75-mm-thick comb (Hoefer). 10. Denaturing PAGE loading solution: 8 M solution of urea in H2 O. 11. Native PAGE loading solution: mixture of glycerin and H2 O (7:3 v/v). 12. Dye mixture: 1 mL formamide, 10 mM Na2 EDTA, pH 8.0, 0.1% (w/v) bromophenol blue, and 0.1% (w/v) xylene cyanol. 13. Stains-All solution: 10% (w/v) solution of Stains-All (Sigma– Aldrich) made in formamide (98%): H2 O (1:1 v/v). 14. Autoclaved H2 O. 2.2. Polyacrylamide Electrophoresis
- 53. 36 Yang et al. 1. Reaction buffer: 10 mM NaH2 PO4 –Na2 HPO4 , pH 7.2. 2. Cu(I) solution: 0.5 mM Cu(CH3 CN)4 PF6 acetonitrile solu- tion (or Cu(NO3 )2 and TCEP·HCl (Tris[2-carboxyethyl] phosphine hydrochloride) 1:2 mixture in water, final CuI concentration 0.5 mM). 1. Solution 1: 40 mg 6-aza-thiothymine (ATT) in 250 mL HPLC-grade acetonitrile. 2. Solution 2: 1.26 mg spermine in 250 mL autoclaved water (25 mM). 3. Mix solutions 1 and 2. Centrifuge briefly since some ATT may not be dissolved. 4. Fucose solution: 8.2 mg fucose dissolved in 1 ml autoclaved water (50 mM). 1. Cyanogen bromide (5 M in acetonitrile, Sigma–Aldrich) (Warning: this is a toxic liquid and care should be taken in the handling of this compound). 2. Reaction buffer: 250 mM morpholineethanesulfonic acid (MES), pH 7.6, and 20 mM MgCl×6H2 O (see Note 3). 3. Powdered dry ice (see Note 4). 4. Two percent (w/v) solution of lithium perchlorate (LiClO4 ) in acetone (spectral grade, Fisher). 5. Sterilized razor blades. 6. Microcon size-exclusion centrifugal filter devices (Millipore, YM 10). 1. Exonuclease VII (ExoVII, source: recombinant, Amersham Biosciences, 1 U converts 1 nmol nucleotide to acid- soluble nucleotide in 30 min at 37°C under standard assay conditions). 2. TAMg (1×) buffer. 1. SPI-1 grade highly orderd pyrolytic graphite (HOPG) (SPI Supplies). Cleave the mica with tape to remove top layer and create a clean surface for sample deposition. 2. Etched silicon cantilevers (OMCL-AC160TS, Olympus). A variety of geometrically well-defined single-stranded DNA templates can be prepared by the following methods. We focus on the preparation of triangular structures T1 and T2 that have 2.3. Metalation of DNA Triangle T1 2.4. MALDI-TOF 2.5. Chemical Ligation 2.6. Enzymatic Digestion of 2D Templates 2.7. Atomic Force Microscopy 3. Methods
- 54. 37 Self-Assembly of Metal-DNA Triangles and DNA Nanotubes with Synthetic Junctions either metal-coordinating ligand 1 or triaryl vertex 2 insertions, respectively. It is of note that these methods can be readily modi- fied to create other 2D shapes (such as squares, pentagons, and hexagons). T1 is composed of three DNA strands (T1a-c), each doubly modified with the dpp ligand 1. These are brought together by the complementary regions of each strand to yield a single-stranded triangle with three preorganized metal-binding pockets (as shown in Fig. 1a). Subsequent metalation can then be characterized by gel electrophoresis, circular dichroism (CD), and thermal denatur- ation experiments. T2 is a triangle made from a linear strand (T2L ) that is cycl- ized via a complementary template (T2P ) and then chemically ligated to the closed product. The single-stranded regions of this polygon allow for further assembly into DNA nanotubes. First, a triangular rung (T2R ) is prepared by the addition of strands R1–3 and L1–3 as shown in Fig. 1b, with sticky ends oriented above and below the plane of the triangle. These rungs can be assem- bled into tubes with the addition of three complementary duplexes Fig.1. (a) Diphenyl phenanthroline (1, inset) inserted into a single-stranded DNA sequ ence. Hybridization of designed sequences into a triangle that template the forma tion of three metal-coordination environments, which can site-selectively bind CuI . (b) Triaryl vertex (2) inserted into a single-stranded DNA template (T2L ) followed by cyclization with cyanogen bromide (CNBr) to yield single-stranded DNA template T2. A triangular rung structure (T2R ) is then assembled from T2 by addition of DNA strands that are designed to provide sticky ends with orientational control.The nanotube is then assembled by connecting the rungs with double-stranded linkers dLS1–3.
- 55. Other documents randomly have different content
- 56. LETTER XIV St. Mary, June 28th, 1842. Rev. Father: Thanks be to God, our hopes have at length begun to be realized; the tender blossom has been succeeded by precious fruit, daily more and more visible in our colony; the chief and people, by their truly edifying conduct, give us already the sweetest consolation. Pentecost was for us and for our beloved neophytes a day of blessings, of holy exultation. Eighty of them enjoyed the happiness of partaking for the first time of the bread of Angels. Their assiduity in assisting during a month at the instructions we gave them, three times a day, had assured us of their zeal and favor; but a retreat of three days, which served as a more immediate preparation, contributed still more to convince us of their sincerity. From an early hour in the morning repeated discharges of musketry announced afar the arrival of the great, the glorious day. At the first sound of the bell a crowd of savages hurried towards our church. One of our Fathers, in a surplice and stole, preceded by three choristers, one of whom bore aloft the banner of the Sacred Heart of Jesus, went out to receive them, and conduct them in procession, and to the sound of joyous canticles, into the Temple of the Lord. What piety—what religious recollection, amidst that throng! They observed a strict silence, but at the same time the joy and gladness that filled their hearts, shone on their happy countenances. The ardent love which already animated 227 these innocent hearts, was inflamed afresh by the fervent aspirations to the adorable Sacrament, which were recited aloud by one of our Fathers, who also intermingled occasionally some stanzas of canticles. The tender devotion, and the profound faith with which these Indians received their God, really edified and affected us. That morning at 11 o'clock they renewed
- 57. their baptismal vows, and in the afternoon they made the solemn consecration of their hearts to the Blessed Virgin, the tutelar patroness of this place.—May these pious sentiments which the true religion alone could inspire, be preserved amongst our dear children. We hope for their continuance, and what increases our hope is, that at the time of this solemnity, about one hundred and twenty persons approached the tribunal of penance, and since that truly memorable occasion, we have from thirty to forty communions, and from fifty to sixty confessions every Sunday. The feast of Corpus Christi was solemnized by another ceremony not less touching, and calculated to perpetuate the gratitude and devotion of our pious Indians towards our amiable Queen. This was the solemn erection of a statue to the Blessed Virgin, in memory of her apparition to little Paul. The following is a brief account of the ceremony. From the entrance of our chapel to the spot where little Paul received such a special favor—the avenue was simply the green sward, the length of which on both sides was bordered by garlands, hung in festoons—triumphal arches, gracefully arranged, arose at regular distances. At the end of the avenue, and in the middle of a kind of repository, stood the pedestal, which was destined to receive the statue. The hour specified having struck, the procession issued from the chapel in this order. At the head was borne aloft the banner of the Sacred Heart 228 followed closely by little Paul carrying the statue and accompanied by two choristers, who profusely strewed the way with flowers. Then came the two Fathers, one vested in a cope, and the other in a surplice.—Finally the march was closed by the chiefs and all the members of the colony emulating each other in their zeal to pay their tribute of thanksgiving and praise to their blessed Mother. When they reached the spot one of our Fathers, in a short exhortation, in which he reminded them of the signal prodigy and assistance of the Queen of Heaven, encouraged our dear neophytes to sentiments of confidence in the protection of Mary. After this address and the singing of the Litany of the Blessed Virgin, the procession returned in the same order to the church. Oh! how ardently we desired that all the friends of our holy religion could
- 58. have witnessed the devotion and recollection of these new children of Mary. It was also our intention not to dismiss them until we had given them the Benediction of the Blessed Sacrament, but unfortunately not possessing a Remonstrance we were obliged to defer this beautiful ceremony until the Feast of the Sacred Heart of Jesus. At that time the Sacred Host was carried in solemn procession, and since then each Sunday after Vespers, the faithful enjoy the happiness of receiving the Benediction. May the blessing of God really descend upon us and our colony. We hope for it through the assistance of your prayers and those of all our friends. I remain, Rev. Father, Your very humble friend and servant, Greg. Mengarini, S.J.
- 59. LETTER XV Fort Vancouver, 28th September, 1841. Reverend Father: Blessed be the Divine Providence of the all-powerful God who has protected, preserved and restored you safely to your dear neophytes. I congratulate the country upon the inestimable treasure it possesses by the arrival and establishment therein of the members of the Society of Jesus. Be so kind as to express to the Reverend Fathers and Brothers my profound veneration and respect for them. I beg of God to bless your labours and to continue your successful efforts. In a few years you will enjoy the glory and consolation of beholding through your means all the savages residing on the head waters of the Columbia, ranging themselves under the standard of the Cross. I do not doubt but that our excellent governor, Dr. McLaughlin, will give you all the assistance in his power. It is very fortunate for our holy religion, that this noble-hearted man should be at the head of the affairs of the honorable Hudson Bay Company, west of the Rocky Mountains. He protected it before our arrival in these regions. He still gives it his support by word and example, and many favors. As we are in the same country, aiming at the same end, namely, the triumph of the holy Catholic faith throughout this vast territory, the Rev. Mr. Demers and myself will always take the most lively interest in your welfare and progress, and we are 230 convinced that, whatever concerns us will equally interest you. The following is an account of our present situation: The Catholic establishment of Wallamette consists of nearly 80 families. The one at Cowlitz of only five,—twenty-two at Nez-quale
- 60. on Puget-sound, which is from 25 to 30 leagues above Cowlitz.[284] Besides these stations we visit from time to time, the nearest Forts where the Catholics in the service of the Hudson Bay Company reside. This is what takes up almost all our time. We are much in want of lay brothers and nuns, of school masters and mistresses. We have to attend to every spiritual as well as temporal affair, which is a great burden to us. The wives of the Canadians, taken from every quarter of the country, cause throughout the families a diversity of languages. They speak almost generally a rude jargon of which we can scarcely make any use in our public instructions—hence proceed the obstacles to our progress,—we go along slowly. We are obliged to teach them French and their catechism together, which occasions much delay. We are really overwhelmed with business. The savages apply to us from all sides. Some of them are indifferent, and we have not time to instruct them. We make them, occasionally, hasty visits, and baptize the children and the adults who happen to be in danger of death. But we have no time to learn their languages, and until now have been without an interpreter to translate the prayers we wish them to learn. It is only lately that I have succeeded in translating them into the Tchinoux language. Our difficulties are greatly increased by this variety of languages; each of the following tribes has a different dialect: The Kalapouyas, towards the head waters of the Wallamette,[285] the Tchinoux of the Columbia river; the Kaijous from Walla-walla; the Pierced Noses, Okanakanes, Flat Heads, Snakes, Cowlitz, the 231 Klickatates from the interior, north of Vancouver;[286] the Tcheheles, to the north of the mouth of the Columbia river; the Nezquales,[287] and those from the interior or of the Puget sound Bay, those of the Travers river, the Khalams[288] of the above mentioned bay, those of Vancouver Island, and those from the northern posts on the sea shore, and from the interior of the part of the country watered by the tributary streams of the Travers river, all have their different languages. Such are the difficulties we have daily to overcome. Our hearts bleed at the sight of so many souls who are lost under our eyes, without
- 61. our being able to carry to them the word of Life. Moreover, our temporal resources are limited. We are but two, and our trunks did not arrive last spring by the vessel belonging to the honorable Hudson Bay Company. We have exhausted our means. The savages, women and children, ask us in vain for Rosaries. We have no more Catechisms of the diocese left to distribute among them; no English Prayer Books for the Catholic Irish; no controversial books to lend. Heaven appears to be deaf to our prayers, supplications and most ardent wishes. You can judge of our situation and how much we are to be pitied. We are in the mean time surrounded by sects who are using all their efforts to scatter every where the poisonous seeds of error, and who try to paralyze the little good we may effect. The Methodists are, first, at Wallamette, which is about eight miles from my establishment; second, near the Klatraps, south of the mouth of the Columbia river; third, at Nez Quali, or Puget-sound; fourth, at the Great Dalles, south of Walla walla; and fifth, at the Wallamette Falls.[289] The Presbyterian Missions are at Wallawalla, as you approach Colville.[290] In the midst of so many adversaries we try to keep our ground firmly; to increase our numbers, 232 and to visit various parts, particularly where the danger is most pressing. We also endeavor to anticipate the others, and to inculcate the Catholic principles in those places where error has not as yet found a footing, or even to arrest the progress of evil, to dry it up at its source. The conflict has been violent, but the savages now begin to open their eyes as to who are the real ministers of Jesus Christ. Heaven declares itself in our favor. If we had a priest to hold a permanent station amongst the savages, the country would be ours in two years. The Methodist Missions are failing rapidly; they are losing their credit and the little influence they possessed. By the grace of God, our cause has prevailed at Wallamette. This spring, Mr. Demers withdrew from the Methodists a whole village of savages, situate at the foot of the Wallamette Falls. Mr. Demers also visited the Schinouks [Chinook], below the Columbia river. They are well disposed towards Catholicity. I have just arrived from Cascades,
- 62. which is eighteen leagues from Vancouver. The savages at this place had resisted all the insinuations of a pretended Minister.[291] It was my first mission, and only lasted ten days. They learned in that time the sign of the cross, the offering of their hearts to God, the Lord's Prayer, the Angelical Salutation, the Apostles' Creed, the ten Commandments, and those of the Church. I intend to revisit them soon, near Vancouver, and to baptize a considerable number. Rev. Mr. Demers has been absent these two months, on a visit to the savages at the Bay of Puget-sound, who have long since besought him to come amongst them. I have not been able to visit since the month of May, my catechumens at Flackimar, a village whose people were converted last spring, and who had turned a deaf ear to a Mr. Waller,[292] who is established at Wallamette. Judge then, sir, how great are our labors, and how much it would advance our 233 mutual interest, were you to send hither one of your Rev. Fathers, with one of the three lay brothers. In my opinion, it is on this spot that we must seek to establish our holy religion. It is here that we should have a college, convent, and schools. It is here that one day a successor of the Apostles will come from some part of the world to settle, and provide for the spiritual necessities of this vast region, which, moreover, promises such an abundant harvest.—Here is the field of battle, where we must in the first place gain the victory. It is here that we must establish a beautiful mission. From the lower stations the Missionaries and Rev. Fathers could go forth in all directions to supply the distant stations, and announce the word of God to the infidels still plunged in darkness and the shadows of death. If your plans should not permit you to change the place of your establishment, at least take into consideration the need in which we stand of a Rev. Father, and of a lay brother, to succor us in our necessities. By the latest dates from the Sandwich Islands, I am informed that the Rev. Mr. Chochure had arrived there, accompanied by three priests, the Rev. Mr. Walsh making the fourth.[293] A large Catholic Church it was hoped would have been ready last autumn for the celebration of the Holy Mysteries. The natives were embracing
- 63. our everlasting faith in great numbers, and the meeting houses were almost abandoned. The Bishop of Juliopolis, stationed at Red River,[294] writes to me that the savages dwelling near the base of the eastern part of the Rocky Mountains have deputed to him a half blood who resides amongst them, to obtain from his Grace a priest to instruct them. Rev. Mr. Thibault is destined for this mission. I remain, Rev. Father, yours, F. N. Blanchet.
- 64. LETTER XVI University of St. Louis, 1st Nov. 1842. Very Rev. Father: In my last letter of August, I promised to write to you from St. Louis, should I arrive safely in that city. Heaven has preserved me, and here I am about to fulfil my promise. Leaving Rev. Father Point and the Flat Head camp on the river Madison, I was accompanied by twelve of our Indians. We travelled in three days, a distance of 150 miles, crossing two chains of mountains,[295] in a section of country frequently visited by the Black Feet warriors, without, however, meeting with any of these scalping savages. At the mouth of the Twenty-five Yard River, a branch of the Yellow Stone, we found 250 huts, belonging to several nations, all friendly to us—the Flat Heads, Kalispels, Pierced Noses, Kayuses, and Snakes. I spent three days amongst them to exhort them to perseverance, and to make some preparations for my long journey. The day of my departure, ten neophytes presented themselves at my lodge to serve as my escort, and to introduce me to the Crow tribe. On the evening of the second day we were in the midst of this large and interesting tribe. The Crows had perceived us from a distance; as we approached, some of them recognised me, and at the cry of the Blackgown! the Blackgown! the Crows, young and old, to the number of three thousand, came out of their wigwams. On entering the village, a comical scene occurred, of which they suddenly made me the principal personage. All the chiefs, and 235 about fifty of their warriors, hastened around me, and I was literally assailed by them. Holding me by the gown, they drew me in every direction, whilst a robust savage of gigantic stature, seemed resolved to carry me off by main force. All spoke at the same time, and appeared to be quarrelling, whilst I, the sole object of all this contention, could not
- 65. conceive what they were about. I remained passive, not knowing whether I should laugh or be serious. The interpreter soon came to my relief, and said that all this uproar was but an excess of politeness and kindness towards me, as every one wished to have the honor of lodging and entertaining the Blackgown. With his advice I selected my host, upon which the others immediately loosed their hold, and I followed the chief to his lodge, which was the largest and best in the camp. The Crows did not tarry long before they all gathered around me, and loaded me with marks of kindness. The social calumet, emblem of savage brotherhood and union, went round that evening so frequently, that it was scarcely ever extinguished. It was accompanied with all the antics for which the Crows are so famous, when they offer the calumet to the Great Spirit, to the four winds, to the sun, fire, earth and water. These Indians are unquestionably the most anxious to learn; the most inquisitive, ingenious, and polished of all the savage tribes east of the mountains. They profess great friendship and admiration for the whites. They asked me innumerable questions; among others, they wished to know the number of the whites. Count, I replied, the blades of grass upon your immense plains, and you will know pretty nearly the number of the whites. They all smiled, saying that the thing was impossible, but they understood my meaning. And when I explained to them the vast extent of the villages inhabited by white men (viz. New York, 236 Philadelphia, London, Paris) the grand lodges (houses) built as near each other as the fingers of my hand, and four or five piled up, one above the other—(meaning the different stories of our dwellings;) when I told them that some of these lodges (speaking of churches and towers) were as high as mountains, and large enough to contain all the Crows together; that in the grand lodge of the national council (the Capitol at Washington) all the great chiefs of the whole world could smoke the calumet at their ease; that the roads in these great villages were always filled with passengers, who came and went more thickly than the vast herds of buffalos that sometimes cover their beautiful plains; when I explained to them the extraordinary celerity of those moving lodges (the cars on the rail road) that leave far behind them
- 66. the swiftest horse, and which are drawn along by frightful machines, whose repeated groanings re-echo far and wide, as they belch forth immense volumes of fire and smoke; and next, those fire canoes, (steamboats) which transport whole villages, with provisions, arms and baggage, in a few days, from one country to another, crossing large lakes, (the seas) ascending and descending the great rivers and streams; when I told them that I had seen white men mounting up into the air (in balloons) and flying with as much agility as the warrior eagle of their mountains, then their astonishment was at its height; and all placing their hands upon their mouths, sent forth at the same time, one general cry of wonder. The Master of life is great, said the chief, and the white men are His favorites. But what appeared to interest them more than aught else, was prayer (religion;) to this subject they listened with the strictest, undivided attention. They told me that they had already heard of it, and they knew that this prayer made men good and wise on earth, and insured 237 their happiness in the future life. They begged me to permit the whole camp to assemble, that they might hear for themselves the words of the Great Spirit, of whom they had been told such wonders. Immediately three United States flags were erected on the field, in the midst of the camp, and three thousand savages, including the sick, who were carried in skins, gathered around me. I knelt beneath the banner of our country, my ten Flat Head neophytes by my side, and surrounded by this multitude, eager to hear the glad tidings of the gospel of peace. We began by intoning two canticles, after which I recited all the prayers, which we interpreted to them: then again we sang canticles, and I finished by explaining to them the Apostles' Creed and the ten Commandments. They all appeared to be filled with joy, and declared it was the happiest day of their lives. They begged me to have pity on them— to remain among them and instruct them and their little children in the knowledge, love and service of the Great Spirit. I promised that a Blackgown should visit them, but on condition that the chiefs would engage themselves to put a stop to the thievish practices so common amongst them, and to oppose vigorously the corrupt morals of their tribe. Believing me to be endowed with supernatural
- 67. powers, they had entreated me from the very commencement of our conversation, to free them from the sickness that then desolated the camp, and to supply them with plenty. I repeated to them on this occasion that the Great Spirit alone could remove these evils—God, I said, listens to the supplications of the good and pure of heart; of those who detest their sins, and wish to devote themselves to His service—but He shuts his ears to the prayers of those who violate His holy law. In His anger, God had destroyed by fire, five infamous villages (Sodom, Gomorrah, 238 etc.) in consequence of their horrid abominations—that the Crows walked in the ways of these wicked men, consequently they could not complain if the Great Spirit seemed to punish them by sickness, war and famine. They were themselves the authors of all their calamities—and if they did not change their mode of life very soon, they might expect to see their misfortunes increase from day to day—while the most awful torments awaited them, and all wicked men after their death. I assured them in fine that heaven would be the reward of those who would repent of their evil deeds and practice the religion of the Great Spirit. The grand orator of the camp was the first to reply: Black Gown, said he, I understand you. You have said what is true. Your words have passed from my ears into my heart—I wish all could comprehend them. Whereon, addressing himself to the Crows, he repeated forcibly, Yes, Crows, the Black Gown has said what is true. We are dogs, for we live like dogs. Let us change our lives and our children will live. I then held long conferences with all the chiefs assembled in council. I proposed to them the example of the Flat Heads, and Pends-d'oreilles, whose chiefs made it their duty to exhort their people to the practice of virtue, and who knew how to punish as they deserved all the prevarications against God's holy law. They promised to follow my advice, and assured me that I would find them in better dispositions on my return. I flatter myself with the hope, that this visit, the good example of my neophytes, but principally the prayers of the Flat Heads will gradually produce a favourable change among the Crows. A good point in their character,
- 68. and one that inspires me with almost the certainty of their amendment, is, that they have hitherto resisted courageously all attempts 239 to introduce spirituous liquors among them. For what is this fire-water good? said the chief to a white man who tried to bring it into their country, it burns the throat and stomach; it makes a man like a bear who has lost his senses. He bites, he growls, he scratches and he howls, he falls down as if he were dead. Your fire- water does nothing but harm—take it to our enemies, and they will kill each other, and their wives and children will be worthy of pity. As for us we do not want it, we are fools enough without it. A very touching scene occurred during the council. Several of the savages wished to examine my Missionary Cross; I thence took occasion to explain to them the sufferings of our Saviour, Jesus Christ, and the cause of His death on the Cross—I then placed my Cross in the hands of the great chief; he kissed it in the most respectful manner; raising his eyes to heaven, and pressing the Cross with both his hands to his heart, he exclaimed, O Great Spirit, take pity on me and be merciful to Thy poor children. And his people followed his example. I was in the village of the Crows when news was brought that two of their most distinguished warriors had fallen victims to the rage and cruelty of the Black Feet. The heralds or orators went round the camp, proclaiming in a loud voice the circumstances of the combat and the tragic end of the two brave men. A gloomy silence prevailed every where, only interrupted by a band of mourners, whose appearance alone was enough to make the most insensible heart bleed, and rouse to vengeance the entire nation. This band was composed of the mothers of the two unfortunate warriors who had fallen, their wives carrying their new born infants in their arms, their sisters, and all their little children. The unhappy creatures had their heads shaven and cut in every direction; they were gashed with numerous 240 wounds, whence the blood constantly trickled. In this pitiable state they rent the air with their lamentations and cries, imploring the warriors of their nation to have compassion on them— to have compassion on their desolate children—to grant them one last favour, the only cure for their affliction, and that was, to go at once and inflict signal vengeance on the murderers. They led by the
- 69. bridle all the horses that belonged to the deceased. A Crow chief mounting immediately the best of these steeds, brandished his tomahawk in the air, proclaiming that he was ready to avenge the deed. Several young men rallied about him. They sung together the war-song, and started the same day, declaring that they would not return empty-handed (viz: without scalps). On these occasions the near relations of the one who has fallen, distribute every thing that they possess, retaining nothing but some old rags wherewith to clothe themselves. The mourning ceases as soon as the deed is avenged. The warriors cast at the feet of the widows and orphans the trophies torn away from the enemies. Then passing from extreme grief to exultation, they cast aside their tattered garments, wash their bodies, besmear themselves with all sorts of colours, deck themselves off in their best robes, and with the scalps affixed to the end of poles, march in triumph round the camp, shouting and dancing, accompanied at the same time by the whole village. On the 29th I bade adieu to my faithful companions, the Flat Heads, and the Crows. Accompanied by Ignatius, Gabriel, and by two brave Americans, who, although Protestants, wished to serve as guides to a Catholic Missionary, I once more plunged into the arid plains of the Yellow Stone. Having already described this region, I have nothing new to add concerning it. This desert is undoubtedly 241 dangerous, and has been the scene of more tragic deeds, combats, stratagems, and savage cruelties, than any other region. At each step, the Crow interpreter, Mr. V. C., who had sojourned eleven years in the country, recounted different transactions; pointing, meanwhile, to the spots where they had occurred, which, in our situation, made our blood run cold, and our hair stand erect. It is the battle ground where the Crows, the Black Feet, Scioux, Sheyennes, Assiniboins, Arikaras, and Minatares, fight out their interminable quarrels, avenging and revenging, without respite, their mutual wrongs. After six days' march, we found ourselves upon the very spot where a combat had recently taken place. The bloody remains of ten Assiniboins, who
- 70. had been slain, were scattered here and there—almost all the flesh eaten off by the wolves and carniverous birds. At the sight of these mangled limbs—of the vultures that soared above our heads, after having satiated themselves with the unclean repast, and the region round me, which had so lately resounded with the savage cries of more savage men, engaged in mutual carnage—I own that the little courage I thought I possessed, seemed to fail me entirely, and give place to a secret terror, which I sought in vain to stifle or conceal from my companions. We observed in several places the fresh tracks of men and horses, leaving no doubt in our minds as to the proximity of hostile parties; our guide even assured me that he thought we were already discovered, but by continuing our precautions he hoped we might perhaps elude their craftiness and malicious designs, for the savages very seldom make their attacks in open day. The following is the description of our regular march until the 10th of September. At day-break we saddled our horses and pursued our journey; at 10 A. M. we breakfasted in a suitable place, that would offer 242 some advantage in case of an attack. After an hour and a half, or two hours' rest, we resumed our march a second time, always trotting our horses, until sunset, when we unsaddled them to dine and sup; we then lighted a good fire, hastily raised a little cabin of branches, to induce our ever watchful foes, in case they pursue us, to suppose that we had encamped for the night; for, as soon as the inimical videttes discover any thing of the kind, they make it known by a signal to the whole party. They then immediately assemble, and concert the plan of attack. In the meantime, favored by the darkness, we pursued our journey quietly until 10 or 12 o'clock at night, and then, without fire or even shelter, each one disposed himself as well as he might, for sleep. It appears to me that I hear you ask: But what did you eat for your breakfast and supper? Examine the notes of my journal, and you will acknowledge that our fare was such as would excite the envy of the most fastidious gastronome. From the 25th of August to the 10th of September, 1842, we killed, to supply our wants, as we journeyed on, three fine buffalo cows, and two large bulls; (only to obtain the tongue and marrow bones) two large deer, as fat as we could have
- 71. wished; three goats, two black-tail deer, a big-horn or mountain sheep, two fine grey bears, and a swan—to say nothing of the pheasants, fowls, snipes, ducks and geese. In the midst of so much game, we scarcely felt the want of bread, sugar or coffee. The haunches, tongues and ribs replaced these. And the bed? It is soon arranged. We were in a country where you lose no time in taking off your shoes; your wrap your buffalo robe around you, the saddle serves as a pillow, and thanks to the fatigues of a long journey of about forty miles, under a burning sun, you have scarcely laid your head upon it before you are asleep. 243 The gentlemen of Fort Union, at the mouth of the Yellow Stone, received me with great politeness and kindness. I rested there during three days. A journey so long and continuous, through regions where the drought had been so great that every sign of vegetation had disappeared, had very much exhausted our poor horses. The 1800 miles that we had yet to travel, were not to be undertaken lightly. After having well considered every thing, I resolved to leave my horses at the Fort, and to trust myself to the impetuous waters of the Missouri in a skiff, accompanied by Ignatius and Gabriel. The result was most fortunate, for, on the third day of our descent, to our great surprise and joy, we heard the puffing of a steamboat. It was a real God-send to us; accordingly, our first thought was to thank God, in all the sincerity of our hearts. We soon beheld her majestically ascending the stream. It was the first boat that had ever attempted to ascend the river in that season of the year, laden with merchandize for the Fur Trade Company. Four gentlemen from New York, proprietors of the boat, invited me to enter and remain on board.[296] I accepted with unfeigned gratitude their kind offer of hospitality; the more so, as they assured me that several parties of warriors were lying in ambush along the river. On entering the boat I was an object of great curiosity—my blackgown, my missionary cross, my long hair, attracted attention. I had thousands of questions to answer, and many long stories to relate about my journey.
- 72. I have but a few words to add. The waters were low, the sand-banks and snags everywhere numerous; the boat consequently encountered many obstacles in her passage. We were frequently in great danger of perishing. Her keel was pierced by pointed rocks, her sides rent by the snags. Twenty times the wheels had been broken to 244 pieces. The pilot's house had been carried away in the tempest; the whole cabin would have followed if it had not been made fast by a large cable. Our boat appeared to be little more than a mere wreck, and in this wreck, after forty-six days' navigation from the Yellow Stone, we arrived safely at St. Louis. On the last Sunday of October, at 12 o'clock, I was kneeling at the foot of St. Mary's Altar, in the Cathedral, offering up my thanksgiving to God for the signal protection He had extended to his poor, unworthy servant. From the beginning of April I had travelled five thousand miles. I had descended and ascended the dangerous Columbia river. I had seen five of my companions perish in one of those life-destroying whirlpools, so justly dreaded by those who navigate that stream. I had traversed the Wallamette, crossed the Rocky Mountains, passed through the country of the Black Feet, the desert of the Yellow Stone, and descended the Missouri; and in all these journeys I had not received the slightest injury. Dominus memor fuit nostri et benedixit nobis. I recommend myself to your good prayers, and have the honor to remain. Your very humble and obedient son in Jesus Christ, P. J. De Smet, S.J.
- 75. EXPLANATION OF THE INDIAN SYMBOLICAL CATECHISM 1. Four thousand years from the creation of the world to the coming of the Messiah. 1843 years from the birth of Jesus Christ to our times. (On the map, each blank line represents a century.) Instruction.—There is but one God; God is a spirit; He has no body; He is everywhere; He hears, sees and understands every thing; He cannot be seen, because he is a spirit. If we are good we shall see Him after our death, but the wicked shall never behold Him; He has had no beginning, and will never have an end; He is eternal; He does not grow old; He loves the good, whom he recompenses; He hates the wicked, whom he punishes. There are three persons in God; each of the three is God—they are equal in all things, c. 2. The heavens, the earth, Adam and Eve, the tree of the knowledge of good and evil, the serpent, the sun, moon, stars, the angels, and hell. Instruction.—God is all powerful; He made the heavens and earth in six days. The first day he created matter, light, the angels. The fidelity of some and the revolt of others. Hell. The second day, the firmament, which is called heavens; the third day, the seas, plants, and trees of the earth; fourth day, the sun, 246 moon, and stars; fifth day, the birds and fishes; sixth day, the animals, Adam and Eve, the terrestrial paradise, and the tree of the knowledge of good and evil. The seventh day was one of rest. A short time after the seventh day, the serpent tempted Eve. The fall of Adam, original sin; its consequences. Adam driven from Paradise, the joy of the Devil. The promise given of a future Saviour, the Son of God. He did not come immediately, but 4000 years afterwards. N. B. It is not well to interrupt too frequently the explanation of the figures on the chart. The necessary remarks on the history of
- 76. religion in general may be made more advantageously apart, and in a continuous manner. Pass at once to the Incarnation of Jesus Christ, the mystery of Redemption, c. 3. Death of Adam. 4. Enoch taken up into heaven; he will return at the end of the world. 5. Noah's Ark, in which four men and four women are saved; all the others perish in the deluge. Instruction.—The history of the deluge. The preaching of Noah. The ark was 450 feet long, 75 wide, and 45 high. Deluge lasts 12 months. The Rainbow. Sem, Cham and Japhet. 6. The Tower of Babel, built by Noah's descendants. Instruction.— About 150 years after the deluge; 15 stories high. Confusion of languages. 7. Abraham, Isaac, Jacob, Joseph, Job, Moses, Aaron, Pharaoh. Instruction.—The history of Abraham, Isaac, Jacob and Joseph. His dreams. He is sold at the age of 16. Jacob passes over to Egypt about 22 years after his son. The Israelites reside in that country 205 years. The history of Moses, the ten plagues of Egypt. The Passover. 247 The Israelites leaving Egypt. The passage of the Red Sea. Pharaoh's army. 8. Sodom, Gomorrah, five cities destroyed by fire from heaven. Lot saved by two angels. Instruction.—Three angels visit Abraham. Two angels go to Sodom. The wife of Lot changed into a pillar of salt. 9. The ten commandments of God given to Moses alone on Mount Sinai. Instruction.—Fifty days after the Israelites have crossed the Red Sea. The promulgation of the Commandments on two tables. First fast of Moses, idolatry of the people, prayer of Moses, golden calf, c. Second fast of Moses. Second tables of the law, 40 years in the desert, the manna, the water issuing from the rock, the brazen serpent. Caleb and Josua. Moses prays with his arms extended. Josua. The passage of the Jordan. Fall of the walls of Jericho. The
- 77. twelve Tribes. Government of God by means of Judges for the space of three to four hundred years. Josua, Debora, Gideon, Jephte, Samson, Heli, Samuel, Saul, David, Solomon, Roboam. Instruction.— The kingdom of Israel formed of ten tribes; it subsisted for 253 years, under 18 kings. That of Juda, formed of two tribes, subsisted 386 years, under 19 kings. 10. The Temple of Solomon. Instruction.—It was built in 7 years. Its dedication. What it contained. It was burned about the 16th year of the 34th age. It was rebuilt at the end of the captivity. This last building was very inferior, and it was at last destroyed forty years after the death of Jesus Christ. Julian, the apostate, was instrumental in accomplishing the prediction of our Saviour. 11. The four great and the twelve minor prophets. 12. Elias taken up into heaven; will return at the end of the world. Eliseus his disciple. Jonas three days in a whale's belly. 248 13. The captivity of Babylon. Instruction.—This captivity lasted for 70 years. It commenced on the 16th of the 34th age, and terminated about 86th of the 35th. 14. History of Susana, Tobias, Judith, Esther. Nabuchodonozer reduced for the space of 7 years to the condition of a brute. The three children in the furnace. 15. The Old Testament. Instruction.—The history of the book of the law, destroyed in the commencement of the captivity. Re-placed at the end of this time by the care of Esdras. Destroyed again under the persecution of Antiochas. 16. The holy man Eleazar. The seven Machabees and their mother; Antiochus, St. Joachim, and St. Anne. 17. Zacharias, Elizabeth, Mary, Joseph. The apparition of the angel Gabriel to Zacharias. Birth of St. John the Baptist. The angel Gabriel appears to Mary. Mystery of the Incarnation of the Word. Fear of Joseph. The visitation. Mary and Joseph leave for Bethlehem.
- 78. Jerusalem is 30 leagues from Nazareth, Bethlehem is 2 leagues from Jerusalem, Emmaus 3 leagues. 18. Jesus Christ, the Son of God, made man for us. The history of the Annunciation. 19. Jesus Christ is born on Christmas day, at Bethlehem. The history of His birth; the angels and shepherds. The circumcision at the end of eight days. The name of Jesus. 20. The star of Jesus Christ seen in the East, predicted by Balaam. 21. The three kings (Magi.) Gaspard, Balthazar and Melchior, having seen the star, come to adore the infant Jesus. Instruction.—The star disappears. The Magi visit Herod. King Herod consults the priests. They point out Bethlehem. The star re-appears. The 249 adoration and presents of the Magi twelve days after our Saviour's birth. 22. Herod wishes to kill the infant Jesus. Herod's fears; his hypocrisy; his recommendation to the Magi. 23. An angel orders the three kings not to return by Herod's dominions, but by another road. The infant Jesus is carried to the temple of Jerusalem forty days after his birth. The holy man Simeon, and the holy widow Anne acknowledge Him as God. This fact comes to Herod's ears; his anger; his strange resolution with regard to the children of Bethlehem, where he thought the infant Jesus had returned. 24. An angel orders Joseph to fly into Egypt with the infant Jesus and Mary his mother. Instruction.—What happened the night after the presentation in the Temple. By the command of Herod all the little children in the town and environs of Bethlehem are put to death. 26. He falls sick and dies at the end of a month, devoured by worms. (Croiset, 18 vol. page 17.)
- 79. 27. An angel orders St. Joseph to carry the infant Jesus, and Mary his mother, back into their own country. They return to Nazareth. 28. Jesus, Mary and Joseph, go up every year to the temple to celebrate the Passover. 29. Mary and Joseph lose the infant Jesus at the age of twelve years, and find him at the end of three days, in the temple, in the midst of the doctors of the law. Instruction.—Fear of Joseph and Mary. Words of his mother. Answer of Jesus. 30. Jesus Christ dwelt visibly on earth for more than 33 years. 31. He taught men the manner of living holily. He 250 gave them the example, and obtained for them the grace to follow it, by his sufferings and death. 32. St. John baptizes Jesus Christ. Instruction.—The birth of the precursor; his life and fasting; his disciples. He declares he is not the Messiah. He points Him out as the Lamb of God. His death. The heavens open at the baptism of Jesus Christ. The Holy Ghost descends. The Eternal Father speaks. Jesus Christ goes into the desert. He fasted for forty days. He is tempted by the devil. The preaching of Christ during three years. His life, His doctrine, His miracles. 33. The twelve Apostles of Jesus Christ—Peter, Andrew, James, John, Philip, Bartholomew, Thomas, Matthew, James, Jude, Simon, Judas. 34. St. Peter, the chief of the Apostles, the Vicar of Jesus Christ on earth, and the first Pope. 35. The Apostles the first Bishops. 36. Judas sells his master for thirty pieces of money. Hatred of the Jews. The treason of Judas.
- 80. 37. Mount Calvary. The cross of Jesus Christ. The other crosses and the robbers. 38. Jesus Christ died on Good Friday. History of the Passion of Jesus Christ. Crucified at 12 o'clock and died at 3. Darkness over the earth. Miracles. Repentance of the executioners. His soul descends into hell. His body is embalmed and laid in the sepulchre, and guarded by Roman soldiers. 39. Jesus Christ rises from the dead on Easter day. History of the Resurrection. He appears to Mary, to St. Peter, to the two disciples going to Emmaus, to the Apostles. Incredulity of St. Thomas. Christ's apparition eight days after. Then also at the lake of Tiberias. The 251 confession of St. Peter. The mission of the Apostles. 40. Jesus Christ ascends into heaven on Ascension day, 40 days after His resurrection. He sends the Holy Ghost to His Church 10 days after His ascension. Wonders and mysteries of the day. 41. He will return to the earth at the end of the world for the general judgment. 42. The seven Sacraments, instituted by our Lord Jesus Christ for our sanctification. The three Sacraments that can be received but once. The five Sacraments of the living. The two of the dead. 43. Prayer in order to obtain the assistance of the grace of God. St. Paul and St. Matthias. 44. Our duties for every day, every week, every month, every year. 45. The six Commandments of the Church. 46. The Church of Constantine the great. 47. The cross of Jesus Christ found on Calvary by St. Helen, after having sought it for three years. The miraculous cross of Constantine. The invention of the Holy Cross. The cross carried by Heraclius in the seventh century. Julian the Apostate.
- 81. 48. The New Testament. The arrangement of the Canon. The discipline ordained by the Council of Nice. 50. St. Augustine converts the English and teaches them the religion of Christ or the Catholic religion. 51. The English follow the religion of Christ, or the Catholic religion, for 900 years. 52. Luther, Calvin, Henry VIII. wander from the way of Christ, reject His religion, that is, the Catholic church. The by-road and its forks represent the Reformation, with its divisions or variations for the last 300 years. The straight road of Jesus Christ existed a long time before. 252 Lucifer or Satan, the first to take a wrong road—he seduces Adam and Eve and their descendants to accompany him. Jesus Christ comes to conduct us into the right road, and enable us to keep it by the grace of redemption. The devil is enraged at the loss he suffers; but he succeeded in the following ages, by inducing men to walk in a new, bad road, that of the pretended Reformation. 53. Arius, Macedonius, Pelagius, Nestorius, Eutyches, Monothelites. 54. Mahomet, Iconoclasts, Berenger, Albigenses, Photius, Wicleff. 55. The four great schisms—of the Donatists, the Greeks, the West, and of England. 56. Luther, Calvin, Henry VIII. 57. Baius, Jansenius, Wesley. 58. The sacred phalanx of the Œcumenical councils. 59. The priests came into the Indian country to teach the Indians the right road or the religion of Jesus Christ, to make them the children of the Catholic church. 60. History of the Catholic missions now flourishing throughout the world.
- 82. FOOTNOTES: [1] Volume xxvii of our series begins with chapter xxxiii of the original New York edition (1838) of Flagg's The Far West. The author is here describing the part of his journey made in the late summer or early autumn of 1836.—Ed. [2] The Vermilion River (which Flagg incorrectly wrote Little Vermilion) rises, with several branches, in the western and southern portions of La Salle County, and flows north and west, entering Illinois River at Rock Island, in Livingston County. Steelesville (formerly Georgetown) is about fifteen miles east of Kaskaskia, on the road between Pinkneyville and Chester; the site was settled on by George Steele in 1810. A block-house fort erected there in 1812 protected the settlers against attacks from the Kickapoo Indians. In 1825 a tread-mill was built, and two years later a store and post-office were erected. The latter was named Steele's Mills. The settlement was originally called Georgetown and later changed by an act of state legislature to Steelesville, being surveyed in 1832.—Ed. [3] Chester is on the Mississippi River, in Randolph County, just below the mouth of Kaskaskia River. In the summer of 1829, Samuel Smith built the first house there, and two years later he, together with Mather, Lamb and Company, platted the town site. It was named by Jane Smith from her native town, Chester, England, and was made the seat of justice for Randolph in 1848.—Ed.
- 83. [4] Flagg is probably referring to Colonel Pierre Menard. See our volume xxvi, p. 165, note 116.—Ed. [5] Philadelphia was founded in 1682. There has been much discussion about the exact date of the founding of Kaskaskia. E. G. Mason was of the opinion that this uncertainty had arisen in the confounding of Kaskaskia with an earlier Indian settlement of the same name on the Illinois River. It seems probable that Kaskaskia on the Mississippi was started in 1699. Consult E. G. Mason, Kaskaskia and its Parish Records, in Magazine of American History (New York, 1881), vi, pp. 161-182, and Chapters from Illinois History (Chicago, 1901); also C. W. Alvord, The Old Kaskaskia Records (Chicago Historical Society, 1906). See also A. Michaux's Travels, in our volume iii, p. 69, note 132.—Ed. [6] The church of the Immaculate Conception, the first permanent structure of its kind west of the Alleghany Mountains, was built in 1720. It was torn down in 1838 and a large brick church built. For a more detailed description of the former, see post, pp. 62-64.—Ed. [7] Hall.—Flagg. [8] Jacques Marquette was a Jesuit missionary, not a Recollect. Consult R. G. Thwaites, Father Marquette (New York, 1902). On Jolliet see Francis Parkman, La Salle (Boston, 1869); and the latest authority, Ernest Gagnon, Louis Jolliet (Quebec, 1902).— Ed. [9] For a short note on the Illinois Indians, consult our volume xxvi, p. 123, note 86.—Ed. [10] Flagg errs in saying that Jolliet published an account of his adventures. His journal was lost in the St. Lawrence River on the return journey. Father Marquette, however, wrote a journal of his travels. See Thwaites, Jesuit Relations, lix, which also contains Jolliet's map of North America (1674).—Ed.
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