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siRNA and Epigenetic
Asma SiddiqueSaloom Aslam
Syeda Zainab Ali
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Topics under discussion Brief History Intro to different terms What is RNAi? What is siRNA? siRNA formation Difference between miRNA and SiRNA siRNA design Therapies Challenges Clinical trials
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History
SiRNA was first discovered by David Baulcombe’s group as part of post-transcriptional gene silencing in plants, in 1993.
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Different terms RNAi siRNA shRNA MiRNA RISC… DICER, ARGONAUTE FAMILY PROTEINS and OTHER
PROTEINS
Off- target effects
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What is RNA interference?
Gene silencing mechanism...siRNA and miRNA
Known as the RNA interference machinery. Once it finds a double-stranded RNA (Dicer), separates the two molecule, cuts it up.
Way to silence genes by preventing the formation of the proteins that they code for.
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Transitive RNAi Organisms have RNA dependant RNA
polymerase that uses the mRNA targeted by the initial anti-sense SIRNA as a template for the synthesis of more siRNA.
These secondary siRNA also target other parts of mRNA.
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When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked:
1. The ribosomes cannot gain access to nucleotides in mRNA
2. Duplex RNA is quickly degraded by ribonucleases
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Double stranded RNA corresponding to a particular gene is a powerful suppressant of that gene.
The suppressive effect of anti sense RNA probably depends on its ability to form dsRNA.
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siRNA
siRNA known as short\small interfering RNA, are a class of 20-25 nucleotide-long RNA molecules that interfere with the expression of genes. It has 2-nt overhangs on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group.
They are produced as part of the RNA
interference (RNAi) pathway by the enzyme Dicer.
They can also be exogenously (artificially) introduced by investigators to bring about the knockdown of a particular gene.
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Sources of siRNA
Plant cells make these from the double stranded RNA of invading viruses.
Scientists make these as agents to turn off the expression of specific genes.
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SiRNA formation Delivery of trigger dsRNA Generation of siRNA pool Capture, unwinding of SiRNA by RISC Binding of SiRNA associated RISC with
target mRNA… ATP dependant Destruction of target mRNA
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SiRNA can also inhibit the transcription of genes:
1. Perhaps by binding to complementary sequences on DNA
2. Perhaps by binding to the nascent RNA transcript as it is formed.
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How these SiRNAs synthesized in the cytosol –gain access to the DNA in the nucleus is unknown.
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miRNA A miRNA (micro-RNA) is a form of single-
stranded RNA which is typically 20-25 nucleotide long.
It is thought to regulate the expression of other genes.
They act by either destroying or inhibiting translation of several mRNA (by binding to a region of complimentary sequences in the 3’UTR of mRNA)
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Studies have shown that miRNAs play a role in the most critical biological events including development, proliferation, differentiation, cell fate determination, apoptosis, signal transduction, organ development, hematopoietic lineage differntiation, host viral interactions and carcinogenesis.
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Effective SiRNA design? Select the target region from the open reading
frame of a given DNA sequence…50-100 nt down stream of the start codon.
Search for sequences 5’AA(N19)UU, in the mRNA sequence and choose those with approx 50% GC content.
BLAST search
Strand incorporation depends upon weaker base pairing…more AT content more incorporation.
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Therapies Synthetic siRNA molecules that bind to gene
promoters can repress transcription of that gene. Repression is mediated by methylation of the DNA in the promoter ; methylation of histones in the vicinity.
Rnai can use as a weapon to counter infections by RNA viruses by destroying their mRNA’s.
Screening genes for their effect on drug sensitivity.
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Why RNA triggers and DNA does not?
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More tightly packed
More stable
RNA is easily hydrolysed
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Challenges of RNAi Finding a vector or delivery system
At what age, a patient should receive treatment
RNAi therapy is long term or only temporary?
Long dsRNA fragments reduce gene expression in mammals
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Clinical trials underway “wet” macular degeneration (targeting VEGF
which encodes vascular endothelial growth factors)
AIDS (targeting an exon used by the HIV envelope protein)
Hepatitis B (targeting four different sequences in the viral genome)
Some cancers
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Any questions???