Biotechnology lecture
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This document provides an overview of RNA interference (RNAi), including its definition, mechanism, generation of small interfering RNA, delivery methods, and applications. RNAi is a conserved mechanism in eukaryotes where double-stranded RNA is cut into small interfering RNA by the enzyme Dicer. These siRNAs then bind to complementary mRNA and induce its degradation via the RNA-induced silencing complex. The document discusses the key components of RNAi, differences between siRNAs and microRNAs, generation of siRNAs, challenges with siRNA delivery, and applications of RNAi technology in areas like cancer treatment, stem cell research, and infectious disease.
Biotechnology lecture
- 1. RNA interference (RNAi) From:- Dr. Abhishek Kumar Mishra Assistant Professor Department of Biosciences Swami Rama Himalayan University 1
- 2. Learningobjectives 2 At the end of the discussion you will be able to learn:- Definition of RNA interference Mechanismof RNAinterference Generation of smallinterfering RNA Smallinterfering RNAdeliverymethods Applicationsof RNAinterference
- 3. 3 RNAsilencing&RNAinterference Several terms are used to described RNA silencing; usually there are three phenotypically different but mechanistically similar phenomena: 1. Cosuppression or post-trascriptional gene silencing (PTGS) in plants 2. Quelling in fungi 3. RNA interference in animal kingdom
- 4. RNAi is a form of RNA based immunity, targeting viruses and genomic repeated sequences RNAi is viewed as a conserved ancient mechanism protecting genomes from nucleic acid invaders The technique has proven effective in Drosophila, Caenorhabditis elegans, plants and in mammalian cell cultures RNAi evolved as a mechanism most likely to protect organisms from viruses modulate transposon activity, eliminate aberrant transcription products RNA interference (RNAi)
- 5. 8 Needfor interference • DefenseMechanism -Defense against Infection by viruses, etc -As a defense mechanism to protect against transposons and other insertional elements • GenomeWide Regulation -RNAi plays a role in regulating development and genome maintenance -30% of human genome regulated
- 6. PTGSin plants: Discoveryof Jorgensenand Napoli(1990) • They were trying to make petunias more purple • Overexpression of petunia gene • Entered homologous RNA Expected: more pigments Observed:white sectors Cosuppression: Loss of mRNAs of both endo-and transgene 9
- 7. Discovery 11 Inject worms with dsRNA corresponding to a gene (important for muscle function) involved in wiggling (unc-22)
- 8. 14 RNAiOverview • During RNAi Double-stranded RNAs cut into short double- stranded RNAs, s(small) i(interfering) RNA's, by an enzyme called Dicer • These then base pair to an mRNA through a dsRNA-enzyme complex. This will either lead to degradation of the mRNA strand • Highly specific process • Very potent activity • So far only been seen in eukaryotes • Evidence 30% of genome is regulated by RNAi
- 9. 15 ThecomponentsIn Interference • RNA -siRNA: dsRNA 21-22 nt. -miRNA: ssRNA 19-25nt. Encoded by non protein coding genome • RISC: -RNA induced Silencing Complex, that cleaves mRNA • Enzymes -Dicer : produces 20-21 nt cleavages that initiate RNAi -Drosha : cleaves base hairpin in to form pre miRNA; which is later processed by Dicer
- 10. Small interfering RNAs (siRNA) • Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of post-transcriptional gene silencing • RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation • A single base pair difference between the siRNA template and the target mRNA is enough to block the process. • Each strand of siRNA has: a.5’-phosphate termini b.3’-hydroxyl termini c.2/3-nucleotide 3’ overhangs 16
- 11. MicroRNA(miRNA) • Originate from capped & polyadenylated full length precursors(pri- miRNA) • Hairpin precursor~70 nt (pre-miRNA) Mature miRNA~22 nt (miRNA) 17
- 12. DifferencebetweenmiRNAand siRNA • Function of both species is regulation of gene expression. • Difference is in where they originate. siRNA originates with dsRNA. • siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target. • miRNA originates with ssRNA that forms a hairpin secondary structure. miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target. • Also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre- transcriptionally (RNA induced transcriptional silencing or RITS) 18
- 13. DICER RNaseIII-like dsRNA-specificRibonuclease Enzymeinvolved in the initiation of RNAi. It is able to digest dsRNA into uniformly sizedsmallRNAs (siRNA). Dicer family proteins are ATP-dependent nucleases. RNAseIII enzymeactsasadimer Loss of dicer→loss of silencing processing in vitro Dicer homologs exist in many organisms including C.elegans, Drosphila, yeast and humans (Dicer isa conserved protein) 19
- 14. Dicer’sdomains 1 4 2 2 Dicer is a Ribonuclease (RNAse III family) with 4 distinct domains: 20 Composed of 4 different domains:- 1.Amino-terminal helicase domain 2.Dual RNAse III motifs in the carboxy terminal segment 3.dsRNA binding domain 4.PAZ domain (110-130 amino-acid domain present in protein like Argo, Piwi..);it is thought to be important for protein-protein interaction 3
- 15. • RISC is a large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA • Unwinding of double- stranded siRNA by ATP independent helicase • The active components of an RISC are endonucleases called argonauteproteins which cleave the target mRNA strand 21 RNA induced silencing complex (RISC)
- 16. Mechanisms of RNA silencing
- 18. Generation of smallinterference RNA 27
- 19. 31 • Effective methods for the delivery of small RNA to allow a sufficient silencing effect in the target organ(s) and/or cells are yet to be developed • In particular, toxicity and side effects of RNAi must be well characterized and limited • Therefore, careful design and selection of target sequence and quantification of the effect on the expression of target protein and mRNA are essential for success of gene interfering approaches siRNA: Delivery
- 20. High-pressure injection 32 • “High-pressure injection” was the first strategy to demonstrate successful delivery of siRNA in vivo • A large volume (1–2mL) of saline containing unmodified siRNA is injected intravenously into the tail vein of mice within very short time (in less than 7 sec), which presumably results in the siRNA molecules being forced into several organs mainly the liver, kidney and to a lesser degree the lung • Certainly, such an approach seems to be impossible in human subjects (1000 mL saline solution containing siRNA per 10 kg of weight)
- 21. 34 • Delivery of siRNA to tissue is a problem both because: – The material must reach the target organ – And it must enter the cytoplasm of target cells • RNA cannot penetrate cellular membranes, so systemic delivery of siRNA is unlikely to be successful • RNA is quickly degraded by RNAse activity in serum and even siRNA chemically modified to be more stable has a half-life of only a few hours at most DeliveryProblems
- 22. 35 • For these reasons, other mechanisms to deliver siRNA to target cells has been devised These methods include: • Viral delivery • Theuseof liposomesor nanoparticles • Bacterial delivery • Chemicalmodification of siRNAto improve stability Solution
- 23. Viraldelivery • Viral delivery has been used extensively in gene therapy to deliver DNA to target cells • There are 5 main classes of viruses used in the delivery of nucleotides to cells: • Retrovirus • Adenovirus • Lentivirus • Baculovirus • Adeno-associated-virus (AAV). 36
- 24. Liposome'sandnanoparticles • Liposomes and nanoparticles have been known as an alternative to viral delivery systems. • Unmodified siRNA has a half-life of less than 1 hour in human plasma and siRNA is rapidly excreted by the kidneys. • Liposomes and nanoparticles can act as envelopes to protect the siRNA from metabolism and excretion, but can also carry specific molecules designed to target the siRNA to specific tissue types • Liposomes such as Lipofectamine have been used to carry siRNA into cells • Nanoparticles such as the cationic polymer, polyethyleneimine (PEI) have also been used to successfully deliver siRNA to target cells 37
- 25. Bacterialdelivery 38 • Bacterial delivery using nonpathogenic bacteria has been used to silence genes in a process known as transkingdom RNA interference (tkRNAi) • Generally, the shRNA is produced in bacteria that invade and release the RNA into eukaryotic cells (hence the term transkingdom) • The bacteria can also be engineered to carry shRNA encoding DNA plasmids • The advantages of this system include: -Safety -Abilityto controlthe vectorusingantibiotics
- 26. Chemicalmodification 39 • Finally, chemical modification of siRNA has been used to improve stability and prevent degradation by serum RNAase • Importantly, these modifications must obviously not affect the RNA interference activity of the siRNA • One of the most common modifications is the use of locked nucleic acidresidues (LNA). • A methylene bridge connects the 4.C with the 2.O in LNA residues. This modification increases the stability of oligonucleotides in serum, without reducing the gene silencing effect
- 28. 43 RNAi in treatment of hematologic disorders result from defective genes RNAi in treatment of Cancers by targeting oncogenes • Improvement efficacy of chemotherapy and radiotherapy • Tumor regression through creation of potentially new mode of chemotherapy RNAi in Stem cell research • Knockout tumor-suppressiongenein mouseembryonicstem cell • Observetumor phenotype • Positive correlation between extent of Trp 53 (suppression gene) inhibition andseverityof disease RNAi in treatment of infectious diseases • RNAi targetstranscriptase&inhibitsviralreplication
- 29. 51 RNAinterferencecharacteristics • dsRNA needs to be directed against an exon, not an intron in order to be effective • Homology of the dsRNA and the target gene/mRNA is required • Targeted mRNA is lost (degraded) after RNAi • ssRNA does not work as well as dsRNA
- 30. 52 Advantageof RNAi • Down regulationof geneexpressionsimplifies"knockout" analysis. • Easierthan useof antisenseoligonucleotides.siRNAmoreeffective and sensitive at lowerconcentration. • Costeffective • HighSpecificity • Middle region9-14 are mostsensitive • With siRNA,the researchercansimultaneously perform experimentsin anycell type of interest • Canbelabelled • Easeof transfectionbyuseof vector
- 31. 53 ImportanceofRNAi • Powerful for analyzingunknown genesin sequenced genomes. • More efforts are beingundertaken to target every human gene via siRNAs • Fasteridentification of gene function • Genetherapy: down-regulation of certain genes/ mutated alleles • Cancertreatments – knock-out of genesrequired for cell proliferation – knock-out of genesencoding keystructural proteins • Agriculture
- 32. THANK YOU 32