By,ISHAQUE P.KBIOCHEMISTRYpondicherry university

si RNA(Small Interfering RNA)

Introduction• Small interfering RNA (siRNA), sometimes known as short

interfering RNA or silencing RNA.

• It is a class of double-stranded RNA molecules, 20-25 base pairs in length with 2-nt at 3’ overhangs.

• siRNA plays many roles, but its most notable is in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequence.

• They are naturally 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.

What Is RNA Interference (RNAi)?

• RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.

• Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling.

• In 2006, Andrew Fire and Craig C. Mello shared the Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm C. elegans, which they published in 1998.

• RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) suppresses expression of a target protein by stimulating the specific degradation of the target mRNA.

• RNAi involves a multistep process.

• The RNAi pathway is found in many eukaryotes and it is initiated by the enzyme Dicer, which cleaves long double-stranded RNA(dsRNA) molecules  into short double stranded fragments of ~20 nucleotides that are called siRNAs.

• Each siRNA is unwound into two single-stranded (ss) ssRNAs, namely the passenger strand and the guide strand. • The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC).• The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex.

The discovery of RNAi

1990 1998 2000 2001 2002 2004 2009

Napoli et al., Plant Cell 2:

279

PTGS in plants (post

transcriptional gene

silencing)

dsRNA leads to silencing in C.elegans

Fire and Mello., Nature 391: 806

dsRNA processed

to siRNAs in RNAi

pathwayZamore et al., Cell 101: 25

siRNAs trigger RNAi

in mammalian

cells

Elbashir et al., Nature 411: 494

siRNA expressed

from vectors

Brmmelkamp et al., Science 296:

550

In vivo siRNA

delivery

Takeshita et al., PNAS 102:

12177

Phase I preclinical

trail

siRNA synthesis• Chemical synthesis.

• In vitro transcription.

• RNase III/DICER digestion of long dsRNA.

General Guidelines1. siRNA targeted sequence is usually 21 nt in length.

2. Avoid regions within 50-100 bp of the start codon and the termination codon.

3. Avoid intron regions.

4. Avoid stretches of 4 or more bases such as AAAA, CCCC.

5. Avoid regions with GC content <30% or > 60%.

6. Avoid repeats and low complex sequence.

7. Avoid single nucleotide polymorphism (SNP) sites.

8. Perform BLAST homology search to avoid off-target effects on other genes or sequences.

Chemical synthesis of siRNA

• Commercial synthesis.

• Expensive process.

• Must screen siRNAs to identify an effective one.

• Synthesis can easily be scaled up.

• siRNAs can be labeled for identification.

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.

RNase III/DICER digestion

• Cocktail of several siRNAs generated by RNase 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.

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.

siRNA Delivery

• Chemical-mediated transfection

- adherent cell lines and some primary cell types• Electroporation

- primary cells, suspension cells, and many difficult to transfected cell lines

• Viral vector

- most all type of cells, in vivo application

- long-term silencing

- adenovirus, lentivirus, retrovirus

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.

Applications……..• RNA interference (RNAi) has become an almost-

standard method for in vitro knockdown of any target gene of interest. From the mechanism, it becomes clear that small interfering RNAs (siRNAs) play a pivotal role in triggering RNAi.

• The use of RNAi for therapeutic purposes holds a great deal of potential for the treatment of viral and genetic diseases, and cancer.

• The specific aim of siRNA therapies is to control gene expression by gene silencing. Many methods are being developed to deliver siRNA to a spe-cific target site in the cell. The delivery methods that hold the most potential include lipo-somes, polymers, chemical modifications, nanoparticles, and aptamers. These delivery me-thods are being used to facilitate therapeutic applications employing siRNA. siRNA can be applied to the treatment of viral and genetic diseases and cancer.

• RNA interference is a vital part of the immune response to viruses and other foreign genetic material, especially in plants where it may also prevent the self-propagation of transposons. Plants such as Arabidopsis thaliana express multiple dicer homologs that are specialized to react differently when the plant is exposed to different types of viruses.

• Although animals generally express fewer variants of the dicer enzyme than plants, RNAi in some animals has also been shown to produce an antiviral response.

• In both juvenile and adult Drosophila, RNA interference is important in antiviral innate immunity and is active against pathogens such as Drosophila X virus. 

• A similar role in immunity may operate in C. elegans, as argonaute proteins are upregulated in response to viruses and worms that overexpress components of the RNAi pathway are resistant to viral infection.

• The role of RNA interference in mammalian innate immunity is poorly understood, and relatively little data is available. Alternative functions for RNAi in mammalian viruses also exist, such as miRNAs expressed by the herpes virus that may act as heterochromatin organization triggers to mediate viral latency.

• Other proposed clinical uses center on antiviral therapies, including topical microbicide treatments that use RNAi to treat infection by herpes simplex virus type 2 and the inhibition of viral gene expression in cancerous cells, knockdown of host receptors and coreceptors for HIV, the silencing of hepatitis A and hepatitis B genes, silencing of influenza gene expression, and inhibition of measles viral replication. Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease.

• The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme.

• Here the Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation.

• The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues.