3-stranded siRNAs

The detailed knowledge about the steps that occur during the natural process of RNAi has inspired scientists to come up with new ways to harness this pathway through the use of novel trigger RNAs. DNA-directed sh-miRs and sh-RNAs, Dicer-substrate RNAs, siRNAs, and single-stranded RNAs can all be used with the aim of obtaining activated RiSC effector complexes containing the guide RNA to seek and destroy target mRNAs.

Now, Bramsen and colleagues report in an Advance Access publication in Nucleic Acid Research on August 28, 2007 on the use of modified 3-stranded siRNAs. The fundamental siRNA design comprises a guide RNA paired with a passenger RNA to form a double-stranded RNA, 21-23 base pairs in length with optimally 2 nucleotide 3’ overhangs. In the case of 3-stranded siRNAs, the passenger strand is split into two separate strands.

3-stranded siRNAs have their roots in stunningly beautiful crystal structures of the Argonaute protein, the Slicer endonuclease of RiSC that cuts the target mRNA, bound to the guide RNA and a complementary RNA (Barford and Patel groups, 2005). In that work, the complementary RNA could have been the target mRNA, but just as well the passenger RNA in an siRNA. Not long after these structures were published, a number of groups reported almost simultaneously that, indeed, the passenger strand of an siRNA is also cleaved. While passenger strand cleavage is not essential for RNAi to work, due to the existence of a bypass mechanism, the Martinez group (Vienna) demonstrated that it greatly stimulates target mRNA cleavage by RiSC. As pre-cleaved, that is 3-stranded siRNAs, could also trigger efficient RNAi, it was concluded that the disruption of the passenger strand allows RiSC to quickly free itself from the passenger RNA for it to act on its real mRNA targets.

The paper by Bramsen et al. now demonstrates and discusses some of the advantages that the use of 3-stranded siRNAs may have. Surprisingly, 3-stranded siRNAs appear to be very forgiving to nucleic acid modifications. RNA modification is considered important to endow siRNAs with the proper drug-like properties, particularly stability, but also reduced off-targeting. Importantly, the authors find that modifications in the guide RNA strand, which is typically more sensitive, tolerated modifications that would abrogate gene silencing within the context of an uninterrupted siRNA. This is especially interesting when it comes to minimising off-targeting by the guide RNA. Small modifications to the second nucleotide from the 5’ end have already been shown to greatly diminish most of the microRNA seed-dependent off-targeting, and the increased flexibility that the 3-stranded siRNA design allows promises further improvements with regard to specificity and also efficacy. Being Danish, the authors suggest the use of locked nucleic acids (LNAs) to achieve this goal. In addition, there is absolutely no off-targeting by the passenger strand as it cannot function any more as a RiSC effector RNA.

You may wonder about the commercial implications of 3-stranded siRNAs. I could imagine the Danish biotech company Santaris to become involved, an oligonucleotides therapeutics company specialising in the use of LNAs. LNAs are highly potent in binding complementary sequences and show great potential for the therapeutic inhibition of microRNAs. Santaris’ patent policy, however, has led to a situation where LNAs are still not very widely used due to their often prohibitively high cost, and for unknown reasons you can almost be assured that when there is an LNA-based publication it involves a group from Denmark.

I would further be interested in whether any of the other academic groups that have contributed to the development of 3-stranded siRNAs claim any IP rights. One of these could be the Zamore group from the University of Massachusetts with ties to CytRx and Alnylam. Martinez on the other hand came out of the Tuschl lab, a co-founder of Alnylam. Lastly, Nastech, a company apparently keen in using anything but the fundamental siRNA design, has applied for a patent involving 3-stranded siRNAs (WO2006US42978 20061103). As often in science, meaningful progress is often made simultaneously by a number of groups and it is almost impossible to determine who deserves ultimate credit.