Short double-stranded RNA duplexes of 20 to 25 nucleotides that program the RNA-induced silencing complex (RISC) to cleave and destroy perfectly complementary messenger RNA targets. Following Andrew Fire and Craig Mello's 1998 discovery of double-stranded RNA interference (RNAi) in nematodes and Sayda Elbashir and Thomas Tuschl's 2001 demonstration that synthetic 21-nucleotide siRNA duplexes silence mammalian genes without triggering interferon toxicity, siRNAs have become both a cornerstone research tool and a class of approved precision medicines.
Class
Short double-stranded regulatory/silencing RNA (RNA interference effector)
Typical Length
21–23 base pairs with 5'-phosphates and 2-nucleotide 3'-dinucleotide overhangs
Subcellular Location
Cytoplasm (P-bodies and rough ER-associated RISC complexes)
Key Complex / Partner
Dicer (RNase III), TRBP, and Argonaute-2 (AGO2) within the catalytic RISC complex
Primary Function
Sequence-specific endonucleolytic cleavage of a single complementary mRNA transcript
Generated endogenously or from viral/exogenous long double-stranded RNA precursors cleaved in the cytoplasm by Dicer, an ATP-dependent RNase III hydrolase that acts as a molecular ruler to excise 21-to-23-nucleotide duplexes.
Each native siRNA duplex carries a 5'-monophosphate and a symmetric 2-nucleotide 3'-hydroxyl overhang on both strands — structural signatures recognized by the PAZ and MID domains of Argonaute proteins.
Synthetic therapeutic siRNAs bypass Dicer cleavage entirely or enter as longer Dicer-substrate duplexes, incorporating 2'-O-methyl, 2'-fluoro, and phosphorothioate backbone modifications that shield the duplex from serum ribonucleases.
Mechanism of Action
During RISC loading, the double-stranded siRNA associates with Argonaute-2 (AGO2); the strand whose 5' end is thermodynamically less stable is retained as the antisense guide strand, while the sense passenger strand is cleaved and ejected. Guided by full Watson–Crick base-pairing across the target mRNA, the RNase H-like PIWI domain of AGO2 ("slicer") hydrolyzes the target phosphodiester bond precisely between nucleotides 10 and 11 opposite the 5' end of the guide strand. The cleaved mRNA fragments lack either a 5' cap or a 3' poly(A) tail and are rapidly degraded by cellular exonucleases, while the intact siRNA-AGO2 complex recycles catalytically across multiple transcript copies.
Biological & Regulatory Roles
Unlike microRNAs, which typically bind with partial seed complementarity to repress dozens of transcripts simultaneously, an siRNA guide requires near-complete complementarity to trigger AGO2 slicing of a single target gene.
In invertebrates, plants, and lower eukaryotes, endogenous siRNAs provide primary antiviral immunity; in human somatic cells, long dsRNA activates innate immune sensors (PKR, RIG-I, MDA5), whereas 21-nt siRNAs evade broad interferon induction.
Interactions & Complexes
Shares the cytoplasmic Dicer and Argonaute machinery with endogenous miRNAs (such as let-7 and miR-21), so supraphysiological siRNA dosing can saturate exportin-5 or RISC loading.
In August 2018, patisiran (Onpattro) became the first siRNA drug approved by the FDA, using lipid nanoparticles to silence mutant and wild-type transthyretin (TTR) mRNA in the liver for hereditary transthyretin amyloidosis.
Triantennary N-acetylgalactosamine (GalNAc) conjugation to the siRNA sense strand enables subcutaneous hepatocyte delivery via the asialoglycoprotein receptor, powering approved therapies such as inclisiran (which silences PCSK9 mRNA to lower LDL cholesterol on a twice-yearly schedule), givosiran (ALAS1), and lumasiran (HAO1). Quantitative target transcript knockdown is routinely monitored via RT-qPCR on thermal cyclers.