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Crook siRNA

Crook siRNA is a modified siRNA-DNA construct that can function as a PCR primer. Jiang et al. in 2005 reported that the "Crook" siRNA-DNA construct functions as a primer for PCR and when transfected into mammalian cells induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart.

Furthermore, the research group suggested that his bifunctional siRNA construct is useful for future in vivo studies on the uptake, distribution, and pharmacokinetics of siRNA.  The application of this type of siRNA-DNA construct could turn out to become quite important for the development of siRNA-based therapeutics and potentially as well as for PCR-based detection of siRNA used for RNAi reverse genetics.


Selective gene silencing by interference RNA or RNAi promised to be a powerful technique for neutralizing or inhibiting gene expression by neutralizing targeted mRNA molecules, however, harnessing this method turned out to be more challenging than anticipated.  https://en.wikipedia.org/wiki/RNA_interference.

In RNA interference (RNAi) double-stranded small interfering RNA (ds siRNA) composed of single-stranded (ss) guide and passenger RNAs are recognized and processed by Ago2 and C3PO, endonucleases of the RNA-induced silencing complex (RISC). When RISC cleaves passenger RNA, the guide RNA is available for base-pairing with its homologous mRNA target. As a result, the degradation of complementary messenger RNA reduces or completely stops gene expression.

Researchers discovered that the 3′ end of passenger RNA could accommodate a DNA extension of 19-nucleotides without the loss of RNAi function. This construct called passenger-3′-DNA/ds siRNA includes a 3′-nuclease-resistant mini-hairpin structure.

Allison and Milner compared different constructs:  Construct (I) contained guide-3′-DNA and ds siRNA, construct (II) included passenger-3′-DNA and ds siRNA, construct (III) contained guide-3′-DNA and ss siRNA, and construct (IV) included passenger-3′-DNA and ss siRNA. The RNAi target selected was SIRT1, a cancer-specific survival factor.

Constructs I–III induced selective knock-down of SIRT1 mRNA and protein in both non-cancer and cancer cells. Apoptotic cell death in the cancer cells was the result. However, construct IV lacking the SIRT1 guide strand was not inducing cell death. Also, adding the 3′-DNA mini-hairpin to the construct made the double-stranded RNA structure resistant to nucleases. In comparison, a similar length DNA extension that did not form a hairpin structure offered little protection to nucleases. Therefore, Allison and Milner suggested that this DNA extension with its mini-hairpin structure, when added to any double-stranded siRNA, protects from degradation by serum nucleases.


Reference

Allison SJ, Milner J.; RNA Interference by Single- and Double-stranded siRNA With a DNA Extension Containing a 3' Nuclease-resistant Mini-hairpin Structure. Mol Ther Nucleic Acids. 2014;2(1):e141. Published 2014 Jan 7. doi:10.1038/mtna.2013.68.

Jiang M, Arzumanov AA, Gait MJ, Milner J.; A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification. Nucleic Acids Res. 2005;33(18):e151. Published 2005 Oct 7. doi:10.1093/nar/gni144.