Small Interfering RNA

04/27/08

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Introduction

Non-coding RNA (ncRNA) is RNA that does not encode a protein, in contrast to mRNA. The well-known ncRNAs include the rRNAs and tRNAs involved in mRNA translation, small nuclear RNAs (snRNAs) involved in splicing and small nucleolar RNAs (snoRNAs) involved in the modification of rRNAs, snRNA and mRNA. Recent discovery of small regulatory ncRNA including microRNA (miRNA), small interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) generates an explosive interest in studies on ncRNA. These small regulatory ncRNAs appear to comprise a hidden layer of internal signals that control various levels of gene expression in physiology and development, including chromatin architecture/epigenetic memory, transcription, RNA splicing, editing, translation and turnover.

RNA interference (RNAi) is an evolutionarily conserved process of posttranscriptional gene silencing (PTGS) induced by double-stranded RNA (dsRNA), leading to the cleavage or degradation of target mRNA with exactly complementary sequence. However, siRNA is 21~23 (18~29)-nucleotide (nt) RNA duplexes with symmetric 2~3-nt 3′- overhangs and 5′- phosphate groups.

Endogenous siRNA is generated by Dicer (an RNAse III family member) from the processing of long dsRNAs, hairpin miRNAs or vector-synthesized short hairpin RNAs (shRNAs). Exogenous siRNA (chemically synthesized) can be converted into active siRNA by an endogenous kinase. The siRNAs associate with cellular proteins to form an RNA-induced silencing complex (RISC), which contains a helicase that unwinds the duplex siRNA in an ATP-dependent reaction. The antisense strand of the duplex siRNA guides the RISC to the homologous mRNA, where the RISC-associated endoribonuclease cleaves the target mRNA at a single site in the centre, which results in the silencing of the target gene.

In 2002, Science journal designated siRNA as “Breakthrough of the Year”. This honor has aroused explosive interest in siRNA studies, leading to exponential increasing number of publications in recent years. In 2006, Nobel Prize in Physiology or Medicine was shared by Professor Andrew Z. Fire at Stanford University, and Professor Craig C. Mello at the University of Massachusetts Medical School for their discovery of siRNA. This honor produced another wave of scientific and industrial interests in siRNA studies.

siRNA movies

How siRNA works

 

General rules for siRNA design
  • siRNA targeted sequence is usually 21 nucleotides in length (19-25, 27-29).
  • Avoid regions within 50-100 bp of the start codon and the termination codon
  • Avoid intron regions
  • Avoid stretches of 4 or more bases such as AAAA, CCCC
  • Avoid regions with GC content <30% or > 60%.
  • Avoid repeats sequence
  • Avoid single nucleotide polymorphism (SNP) sites
  • Perform BLAST homology search to avoid off-target effects on other genes or sequences
  • Match various species if possible
  • low internal stability of the sense 3'end; at least one A/T duplex between position 15-19
  • A at position 3, U at position 10, G at position 13, A at position 19 (sense strand) and absence of G at position 19
  • To minimize off-target effects, use oligos with at least 3 mismatches to unrelated sequences.

siRNA design programs

 

 The RNAi Web

 

TUSOM Neuroscience SFNAGADDWASBMB


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This site was last updated 04/27/08