siRNA and Epigenetic

Asma SiddiqueSaloom Aslam

Syeda Zainab Ali

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

History

SiRNA was first discovered by David Baulcombe’s group as part of post-transcriptional gene silencing in plants, in 1993.

Different terms RNAi siRNA shRNA MiRNA RISC… DICER, ARGONAUTE FAMILY PROTEINS and OTHER

PROTEINS

Off- target effects

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.

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.

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

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.

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.

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.

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

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.

How these SiRNAs synthesized in the cytosol –gain access to the DNA in the nucleus is unknown.

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)

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.

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.

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.

Why RNA triggers and DNA does not?

More tightly packed

More stable

RNA is easily hydrolysed

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

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

Any questions???