Oligoengineer Blog

The regulation of microRNA is exciting and I certainly like working with customers around the world who are making very interesting discoveries that will someday lead to new medicines.

However, when I hear about modified RNA antagonists, LNA antagonists, or any other AON-like approach, I wonder why this time it will be any different than in the past. After all, didn’t this same approach prove non-specific and sometimes toxic in the 90’s? Heavily modified RNA or modified oligos may escape exonuclease degradation, but the mechanism is ‘antagonism’ and that doesn’t require a perfect nucleic acid complement to bind. Is the industry repeating itself?

I’m guessing that, like siRNA, there will be a ‘therapeutics moment’ when just working isn’t going to be enough. And again, the antagonism approach will fail. MicroRNA genes share a lot of common 22 nt motifs and it won’t take much research to see that antagonists interfere with many semi-complementary microRNA. Reduce these targets down to just ‘mature’ regions, and I can only guess it gets worse. Increase the Tm, base pairing 20 of 22 nt gets even worse.

For us, we prefer our newer approach based on specifically structured RNA. It’s pure RNA. It’s not toxic or reliant on chemical modifications. It’s double-hairpin structure provides it’s novel mode of action by endonuclease recruiting but it just so happens to help it resist degradation- making it ideal as a therapeutic.  Most importantly, the mechanism is catalysis of the target gene- activated upon perfect duplex formation by recognition of the target site. In the case of microRNA, its 22 nt that acts as a guide for cleavage. Unlike antisense, this structured RNA recruits and guides an RNase/RNA complex to a target motif. Upon hybridization, the target is cut. It’s neither antisense or siRNA, but the best of each.

For those of you interested in novel modes of gene modulation, I welcome you to give it a try. For targeting microRNA genes, it’s called X-miR. X-miR are available at Oligoengine as either synthetic RNA or as clones for making stables.

I nearly forgot we had a blog. It’s been very busy at Oligoengine. New changes to note:

1. Oligoengine is now part of Halo-Bio RNAi Therapeutics. Research-to-therapeutics support is available to all those seeking to commercialize their research using A-RNAi.
2. The full X-miR microRNA Repressor libraries are now available.
3. All dsRNA based approaches have been replaced with improved counterparts.
4. New software to access 11,000 new products and design tools for new technologies are forthcoming.

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.

Now that Adobe has finally released Shockwave 11 for Intel Macs, we will begin to finish development of Oligoengine 3.0. New features will include collaborative projects, multi-session logins, saved states, real-time oligo catalogs for your experiment, and a wealth of new RNAi technologies. Please note that the interface of Oligoengine 3.0 is significantly different than the current 2.0 version. For all of us using Intel macs, here is the download link: DOWNLOAD SHOCKWAVE 11

The pSUPER section is updated and now back online. The protocols, maps, and sequences are easily downloaded from within the page as well. I appreciate the emails lately and I’m glad to get your support data back online.

It’s time to redo everything! Please pardon my dust as I update the Oligoengine website. I just couldn’t stand it any longer. Over the next few weeks, I will be posting new content, updated information, and posting new products for RNAi.