Hi Atul,

My understanding is that we do this because of the way the RISC complex is assembled.  Typically, Ago proteins are loaded with a double-stranded siRNA:siRNA* hybrid, then the non-targeting strand (we typically call it the siRNA* strand) is cleaved by Ago's endonuclease activity and released, creating the mature, mRNA-targeting Ago:siRNA complex.  As far as I know, Ago proteins cannot be loaded with a single-stranded RNA, only a double-stranded RNA (and, specifically, one of 20-ish base pairs with a 2nt, 3' overhang).

Hope that makes sense!

Cheers,

Dan

Hi Atul,

My understanding is that we do this because of the way the RISC complex is assembled.  Typically, Ago proteins are loaded with a double-stranded siRNA:siRNA* hybrid, then the non-targeting strand (we typically call it the siRNA* strand) is cleaved by Ago's endonuclease activity and released, creating the mature, mRNA-targeting Ago:siRNA complex.  As far as I know, Ago proteins cannot be loaded with a single-stranded RNA, only a double-stranded RNA (and, specifically, one of 20-ish base pairs with a 2nt, 3' overhang).

Hope that makes sense!

Cheers,

Dan

To add further to Daniel's answer:

There were studies using single anti-sense strand siRNA aiming to reduce sense strand induced off-target effects, however, their bioavailability and the RNAi activity were greatly reduced due to faster single strand degradation. Double stranded siRNAs with overhangs have been proved for their enhanced half-life and RNAi effect. 

Longer siRNAs more than 20-21 bases, like DssiRNAs are also used for prolonged and enhanced RNAi effect as their intracellular bioavailability is higher than 21-mer siRNAs. However, the cost of synthesis and purification of such long siRNAs make it more expensive (so people tend to use shRNA vectors encoding longer DssiRNAs).

Hi, Thanks for the nice answers. My understanding for the subject was that its "Dicer" which specifically binds with dsRNA's and thus cleaves-off them to small (20-22nt) siRNAs, which still double stranded and then loaded/transferred to Ago2 protein of RLC (RISC loading complex). The RLC (Ago2, an RNase) uncouples the strands based on stereochemistry or stability (5' end). Thus finally single strand being generated and associated with Ago2 matches with the mRNA sequences based on homology and thus allowing the Ago-2 to chew up (endonuclease activity) the target mRNA's of respective genes. But I was wondering does ssRNA (sense or antisense) directly can be loaded to RLC complex without getting degraded so we can avoid one step i.e. Dicer binding/recognition of the dsRNA's? Sometimes, timely non-upregulation of Dicer protein expression also poses challenge into some model organisms to validate the RNAi protocol. 

Both of your answers were quite helpful to my understanding to the subject. Thanks a lot!!!