As the RNAi pathway is being rapidly dissected structurally, RNAi triggers continue to evolve rapidly both in terms of safety and efficacy. The 5’ end of the guide strand of an siRNA has received particular interest in that regard, and a paper in Nature now provides structural support for why human Argonaute 2, the important Slicer enzyme that cuts target mRNAs, preferentially accommodates ‘Us’ (uracil) and ‘As’ (adenine) of the genetic alphabet in its 5’ guide binding pocket (Frank et al.: Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2).
Humans contain 4 related Argonaute proteins, Ago1-4, but only Ago2 can function in RNAi gene silencing. Not only will good incorporation efficiencies of RNAi triggers in Ago2 enhance gene silencing, loading less into Ago1, 3, 4 which only contribute to microRNA off-targeting without adding to RNAi efficacy should also improve the safety of RNAi Therapeutics. In fact, they may even compete with loaded Ago2 for target mRNAs and thus inhibit RNAi gene silencing. Similarly, because the RNAi function is pretty much dispensable in most cell types and developmental stages, burdening only Ago2 with the RNAi trigger should also reduce the risk of saturating the microRNA gene silencing functions of cells as Ago1, 3, and 4 should be able to cover for Ago2’s contribution in microRNA gene silencing through genetic redundancy.
Because of the structural relatedness of Argonautes, however, many RNAi triggers typically end up to some degree in all 4. In theory, however, Ago1-4 will differ in molecular detail and this provides the rationale for attempting to design RNAi triggers in a way (for example through chemistry, the nature of the double-strand RNA structure, or directing them into certain processing pathway) that the guide strand of an RNAi trigger gets preferentially loaded into Ago2. The 5’ end is of particular interest here because this is where the guide strand exhibits most ‘personality’ by exposing not only the relatively anonymous sugar phosphate backbone, but the entire nucleotide including its 5’ modification, typically a phosphate, to the Argonaute proteins.
It is therefore not surprising that, as indicated also by the patent literature, companies in the space are working on 5’ strategies that at the moment are particularly aimed at increasing the affinity of the guide strand towards Ago2. The message from the present Nature paper is apparently quite simple in that it suggests uracils and, a little bit behind, adenines to give you the best affinities. This is also consistent with the observation that the majority of microRNAs, the naturally occurring small silencing RNAs, also bear a uracil at their 5’ ends. An important technical advance in that paper is that these conclusions were drawn from studying the relevant domain (‘MID domain’) of human Ago2, including its crystal structure, while most of the earlier structure-based analyses have made use of more distantly related bacterial Argonaute proteins.
Related observations have been reported last year, when it was found that replacing the 5’ uracil of the microRNA let-7 with another nucleotide dramatically reduced its RNAi activity (Felice et al: The 5' terminal uracil of let-7a is critical for the recruitment of mRNA to Argonaute 2). Interestingly, the effect did not seem to be so much on the small RNA by Ago2, but rather at the target mRNA recognition step. It should be noted here that the present Nature studies only looked at the structure and affinities of single nucleotides, not the entire guide strand, with Ago2.
For the future, I would hope, for the noted potential for enhanced efficacy and especially specificity to see more of these studies, particularly those that systematically investigate the effect of the 5’ nucleotide on the relative incorporation efficiencies of silencing RNAs into the 4 human Argonautes. These would complement other studies, including one to which I contributed, that look at the secondary structure of the RNAi trigger with respect to the various Argonaute incorporations. A nice precedence of differential Argonaute loading based on the nature of the 5' nucleotide already exists in plants where it has been found that the mere identity of the 5’ nucleotide determines the sorting of a small silencing RNA into the various Argonautes.
Whether a 5’ uracil has discrimination effects in addition to the affinity benefit remains to be seen. However, the results already suggest that since the 5’ end of a small silencing RNA does not directly interact with the target mRNA anyway, it might be worth thinking about siRNA screening libraries that all start with a uracil. Of potentially even more immediate importance, DNA-directed RNAi promoter strategies ought to be re-considered as many of these still employ minimal hairpin RNAs driven from Pol-III promoters in which the guide strand is found at the 5’ end of the hairpin and that initiate with a non-uracil residue.
If you are a non-scientist and still reading this, you are doing pretty well. The take-home message is that while very potent, picomolar RNAi triggers can already been routinely discovered, RNAi trigger design is still an area with lots of potential for further improvements. In 5-10 years I can imagine RNAi Therapeutics to have matured to an extent where structurally highly adapted RNAi triggers effect potent gene silencing with minimal off-targeting activity.