Controlled polymerase ribozyme

[Bryan Bishop]

My perspective is that a controlled polymerase is useful regardless of whether it makes DNA or RNA. Instead of a protein-based polymerase, I have been thinking about the prospects for controlling an RNA-based RNA polymerase ribozyme, which might be easier than making a controlled (protein) polymerase.

These are "easy" to make (SELEX). It's the same technique used for selecting aptamers and DNAzymes.

There already exist a few synthetic RNA polymerase ribozymes. They were investigated because of "RNA world hypothesis" reasons and "origin of life" reasons. Consequently the investigators were more interested in proof-of-concept behind template-dependent RNA polymerase. Their best polymerase only polymerized from a 10 nt template and only polymerized up to ~90 nt. However I think that could be improved through additional rounds of SELEX.

In particular for our purposes we don't need the template-dependent activity. All that we need is random incorporation of nucleotides into the RNA strand. Our SELEX selection criteria would be the total length of the output synthesized RNA, which is trivial to test for. Also I think there are already some ribozymes that create long branched RNA and branched DNA molecules.

What about control over the ribozyme? Well, for DNAzymes there are azobenzene methods for making DNAzymes photo-switchable. For ribozymes, there are riboswitches, and also you can physically attach aptamers that could bind to other physical components that could materially impact the polymerase's behavior.

for example- http://pubs.acs.org/doi/abs/10.1021/sb300120n

and for photoswitching to create conformational change, http://pubs.acs.org/doi/abs/10.1021/ar400308f

Unrelated, but it turns out that DNA origami is electrically conductive:

https://www.researchgate.net/profile/Jinglin_Kong/publication/271525802_Ionic_Conductivity_Structural_Deformation_and_Programmable_Anisotropy_of_DNA_Origami_in_Electric_Field/links/5610fffe08ae0fc513f1a33e.pdf

There's no in vitro protein synthesis step, so it's a much faster machine to evolve. I think light would be the ideal control method, you would need one color for each nucleotide, then an additional color for "perform the next incorporation and then pause" (e.g. the idea would be after incorporation of a nucleotide, the ribozyme would lock up until signaled to perform the next incorporation event).

Ideally there would be a genetically-encodable method of photoswitching a ribozyme, which would mean you could do lots of random RNA in a large batch until you find something that responds somewhat correctly to light stimulation. I think an aptamer method makes sense, you could bind to some photo-switching vitamin molecule and incorporate that as an external element into the design of the ribozyme after synthesis of the RNA (and probably after initial folding if any folding).

more on this subject:
http://gnusha.org/logs/2016-07-14.log

http://gnusha.org/logs/2016-07-15.log

- Bryan
http://heybryan.org/
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