SiRNA
- 1. R O S H N I . P M S C B I O C H E M I S T R Y SiRNA : structure and function
- 2. What is RNA? Ribonucleic acid Ribonucleotides (Ribose, base, & phosphate) Types Coding: messenger RNA (mRNA) Non-coding: Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA) Small nucleolar RNA (snoRNA) Interference RNA (RNAi) Short interfering RNA (siRNA) Micro RNA (miRNA)
- 3. siRNA : (small-interfering RNAs) Function of the species is regulation of gene expression siRNA originates with dsRNA Approximately 21-28 nucleotides in length. siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target
- 4. siRNAs have a defined structure 19 nt duplex 2 nt 3’ overhangs
- 5. siRNAs found in nature are derived from the cytoplasmic processing of long dsRNA by the RNase-IIItype enzyme termed Dicer.Dicer cleaves long dsRNA into 21- to 28- nucleotide siRNA duplexes that contain 2-nucleotide 3′ overhangs with 5′ phosphate and 3′ hydroxyl termini. Components of the RNAi machinery specifically recognize the siRNA duplex and incorporate a single siRNA strand into a protein complex termed the RNA-induced silencing complex (RISC). RISC cleaves mRNAs containing perfectly complementary sequences, 10 nucleotides from the 5′ end of the incorporated siRNA strand.
- 7. siRNA design
- 8. Function of SiRNA Function of the species is regulation of gene expression : (RNAi)
- 10. Shooting mRNA means RNA interference
- 11. RNAi: two phases Initiation Generation of mature siRNA Execution Silencing of target gene Degradation or inhibition of translation
- 12. How does RNAi work?
- 13. •RNAi is mediated by small interfering RNAs (SiRNAs) that are generated from long dsRNA. •Long dsRNAs are cleaved by a ribonucleaseIII (RNaseIII) type protein Dicer. •Dicer homologues can be found in S.pombe, C.Elegans, Drosophila, plants, and mammals, suggesting that small RNA – mediated regulation is evolutionarily ancient and may have critical biological roles.
- 14. DICER Dicer (class 3 RNAse III) cleaves long dsRNA into siRNA 21-25nt dsRNA Symmetric 2nt 3’ overhangs, 5’ phosphate groups
- 16. Dicer cuts dsRNA into short RNAs Vanhecke, D.; Janitz, M. Drug Discov. Today, 2005, 10, 205-225. Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.
- 19. How do Dicer proteins work in dsRNA processing? The PAZ (Piwi Argonaute Zwille) and RNase III domains play central roles in excising siRNAs preferentially from ends of dsRNA molecules.
- 20. PAZ domains are shared with Argonaute proteins and are specialized to bind RNA ends, especially duplex ends with short (2 nt) 30 overhangs. An end engages the Dicer PAZ domain, and the substrate dsRNA then extends approximately two helical turns along the surface of the protein before it reaches a single processing center.The center resides in a cleft of an intramolecular dimer involving the RNase III domains. Each of the two RNase III active sites cleaves one of the two strands, leading to staggered duplex scission to generate new ends with 2 nt 30 overhangs. The reaction leaves a 50 monophosphate on the product ends, consistent with a requirement for this group during later stages of silencing.
- 22. processing the dsRNA into 21-23 nt fragments 34 27 21 20 16 short-interfering RNA Q uic kTime ™ a n d a G IF d ec o mp re s so r a re n ee d ed to s ee this p ic tu re . Tuschl, 2001
- 23. RNAi silencing complex • may be associated with translating ribosomes • active RNAse enzyme not yet identified • may participate in endogenous pathways that silence genes via translational repression
- 24. RNA Induced Silencing Complex (RISC) RNAi effector complex Preferentially incorporates one strand of unwound RNA . Antisense How does it know which is which? The strand with less 5’ stability usually incorporated into RISC.
- 26. Argonaute proteins lie at the heart of RISC
- 27. the antisense strand of the siRNA guides cleavage
- 28. Basic research Determining protein function Easier than a knockout and may be used for partial knockdowns Clinical research Cancer, hypercholesterolemia, infections, developmental defects
- 29. siRNA for treatment of AMD Age-related macular degeneration (AMD) is an eye disease that destroys central vision by damaging the macula, the central region of the retina. AMD affects millions of people worldwide. The main symptoms of AMD is dim or fuzzy central vision. Objects may appear distorted or smaller then they really are, and straight lines may appear wavy or curved. Patients may develop a blank or blind spot in their central field of vision. There are no effective therapies.
- 30. siRNA for treatment of AMD AMD is often associate and promoted by neovascularization - new blood vessel growth. Macular neovascularization is stimulated by interaction of vascular endothelial growth factor (VEGF) with vascular endothelial growth factor receptor VEGFR-1. Inhibiting production of VEGFR-1 should stop neovascularization and prevent development of AMD. Inhibition of blood vessel growth in corneas of mice after a single intravitreal injection
- 31. siRNA targeting ApoB ApoB is a liver enzyme essential for the assembly and secretion of low-density lipoprotein (LDL), which are required for metabolism of cholesterol. High levels of ApoB and LDL increase risk of coronary artery disease. Alnylam Pharmaceuticals developed siRNA to target ApoB. They first used chloresterol-conjugated siRNA to demonstrate its effect on ApoB production in mice.
- 33. HIV levels can be reduced by 30-50 fold by siRNA!!!
- 36. SiRNA have become not an exciting new tool in nolecular biology but also the next frontier in molecular medicine. Significant hurdles remain, most notably guaranteeing specificity and finding safe and efficacious delivery system. Work is ongoing to solve these problem, but the therapeutic promise of SiRNA remains great. CONCLUSION