SiRNA

Detail study about siRNA

1. Presentation on: siRNA Presented by: Kashikant Yadav M pharm (Pharmacology)

2. Introduction Also as short interfering RNA or silencing RNA Is a class of double-stranded RNA molecules 20-25 base pairs in length similar to miRNA operating within the RNA interference ( RNAi ) pathway by the enzyme Dicer

3. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, resulting in no translation.

4. RNA Interference (RNAI) Biological process in which RNA molecules inhibit gene expression ,typically by causing the destruction of specific mRNA molecules. Phenomenon in which dsRNA suppresses expression of a target protein by stimulating the specific degradation of the target mRNA

5. siRNA synthesis Chemical Synthesis In vitro transcription RNAase III/DICER digestion of long dsRNA

6. Chemical Synthesis Commercial synthesis. Expensive process. Must screen siRNAs to identify an effective one. Synthesis can easily be scaled up. siRNAs can be labeled for identification.

7. Best for: Studies that require large amounts of a defined siRNA sequence Not suitable for: Screening siRNA sequences Long term studies

8. In vitro transcription of siRNA In vitro transcribe sense and antisense RNA strands from dsDNA template; hybridized RNA strands to create siRNAs . Fast turn around. Lower concentration. Must screen siRNAs to identify an effective one. siRNAs can be labeled.

9. RNAase III/DICER digestion Cocktail of several siRNAs generated by RNAase III/Dicer digestion of long dsRNA Leaves same overhang characteristics No need to screen for effective siRNA SiRNA cocktail can be labeled Does not identify single effective siRNA sequence Non-specific effects

10. siRNA cocktails made with RNase III Complementary RNA strands (100-500nt) transcribed from dsDNA template and then hybridized to long dsRNA DNase and RNase used to remove DNA template and unhybridized RNA strands RNase III digests dsRNA into population of 12-15mer dsRNA that fuction as siRNAs Clean up.

11. Significance of the RNAi RNAi protects against viral infection. RNAi secures genome stability by keeping mobile elements silent. RNAi -like mechnisms repress protein synthesis and regulate the development of organisms. RNAi -like mechanisms keep chromatin condensed and suppress transcription. RNAi offers a new experimental tool to repress genes specifically. RNAi might be a useful approach in future gene therapy.

12. applications Testing Hypotheses of Gene Function Target Validation Pathway Analysis Gene Redundancy Functional Screening siRNAs as Therapeutics