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

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

7. 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.

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

9. 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.

11. 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

12. 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.

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

15. applications
i) Testing Hypotheses of Gene Function
ii) Target Validation
iii) Pathway Analysis
iv) Gene Redundancy
v) Functional Screening
vi) siRNAs as Therapeutics