Rethinking gene silencing, making Asymmetric RNAi.
April 11th, 2008
The use dsRNA, whether a synthetic siRNA duplex or pre-cursor shRNA, present symmetrical RNA duplexes to gene silencing machinery. The industry defines such duplexes by ‘template and guide’ strand. Most RNAi designers focus on the guide portion of the dsRNA duplex and ensure ‘guide’ complimentarity to a target gene. But, what happens when the symmetrical dsRNA loads the 3′ end of the template strand? In terms of hydrogen bonds, what’s the difference? It seems logical to assume there are none. One can even flip template for guide design methods and silence a target gene either way. Some reports suggest duplex thermodynamics control loading. But there are thousands of effective siRNA contrary to the thermodynamic description. Are the claims of that duplex thermodynamics controls loading direction reliable enough for precise applications?
This is an oversimplification, but we feel it illustrates a long-standing challenge with dsRNA based RNAi tools and potential therapeutics. For several years, we have been working on improving gene silencing. We have programmed new and rational design software for RNAi, invented chemically asymmetric siRNA duplexes for specificity, and now dreamt up a new class of RNAi precursors that bring stable forms of RNAi the specificity that was previously exclusive to chemically modified siRNA. This is done with the use of a novel RNA precursors. We are formalizing this entire approach as ’Asymmetric RNAi’ (A-RNAi), since it chemically removes the symmetry in dsRNA that can lead to sense-strand mediated off-target suppression.
Also, we have defined 3 new precursors that can used in place of classical shRNA, siRNA, or antisense for those who wish to practice A-RNAi. The precursors versatile and can be expressed constitutively from pSUPER or transfected as synthetic RNA in transient applications. To make things simple, these new forms are named similar to the classical counterparts; A-shRNA, A-siRNA, and A-antisense.
Each new Asymmetric precursor has purposeful design properties that suggest unique traits as gene silencing molecules. These traits may even go further than the specificity goal of their design. For example, A-siRNA can be constitutively expressed as RISC guiding molecule that should not involve or overload Dicer. Will that lead to cleaner gene silencing signatures? Less interferon response? Also, they are resistant to degradation in serum making them useful for invivo applications. Well, this is beyond the scope of this blog and we’ll leave it up to researchers around the world to dive into the intricacies of Asymmetric RNAi applications.
For now, I’m just pleased to introduce this approach to you and make it available at Oligoengine. We invite you to review and consider Asymmetric RNAi in your research. Feel free to email me if you have questions.