Thread by @Niklas_Freund1

Proud to announce that my first first-author paper is online! We (@Taylor_Lab_Cam, @PhilHolliger and our co-workers) demonstrate how a novel engineered polymerase unlocks efficient synthesis and evolution of therapeutic 2'-modified nucleic acids.

nature.com/articles/s4155…

🧵(1/n)

Almost twenty nucleic acid therapeutics have been approved for use in the clinic. Antisense oligos are synthesised by solid-phase synthesis, which is efficient but costly, limited to short oligomers and hard to scale up to large numbers of sequence or chemistry variants. (2/n)

Discovery might be accelerated by tools for the rapid synthesis of many variants: polymerases. Also, for antisense therapeutics (obvious sequence), rational design is feasible, but for therapeutic modalities like aptamers, polymerases enabling in vitro evolution are needed. (3/n)

The activity of many nucleic acid drugs depends on 2'-modified nucleotides conferring high Tm and nuclease resistance: 2'-O-methyl (2'OMe), which has attracted much attention, and the bulkier 2’-O-(2-methoxyethyl) (MOE), which had been impossible to synthesise enzymatically.(4/n)

We set out to engineer a polymerase enabling synthesis of 2’-mod RNA by identifying steric clashes between nascent strand 2’OMe and polymerase residues. When mutated to smaller amino acids, two residues showed a striking synergistic increase in 2'-mod RNA synthesis activity.(5/n)

Interestingly, the two identified gatekeeper residues (T541 & K592) are part of previously-described motifs that are highly conserved in polB polymerases across all phyla. (6/n)

Mutating this nascent strand steric gate in the double mutant 2M enables efficient 2’OMe- & MOE-RNA synthesis and unlocks in vitro evolution experiments. In a pol blend, 2M enables mixed 2'OMe-RNA/LNA library synthesis: @Taylor_Lab_Cam @JackHervey pubs.rsc.org/en/content/art… (7/n)

@Taylor_Lab_Cam performed the in vitro evolution of the first all-2’OMe-RNA enzymes, selected to cleave RNAs with single-nucleotide discrimination, allowing for the allele-specific cleavage of oncogenic mutant mRNA of KRas and β-catenin while leaving wild-type allele intact.(8/n)

To show whether MOE-RNA is a viable scaffold for aptamers, we modified an existing 2’OMe-RNA VEGF-aptamer with MOE. A 50% substitution variant still bound VEGF with identical affinity compared to the parent aptamer. This suggests the first MOE-RNA aptamer is within reach. (9/n)

In summary, sterically demanding 2’-modified nucleic acids used in antisense drugs have been challenging to synthesise enzymatically. Mutation of two gatekeeper residues in an archaeal polymerase unlocks efficient synthesis of 2'OMe- & MOE-RNA and evolution of 2’OMezymes. (10/n)

Further reading in an accessible style:

Insight on Research | MRC LMB Website: www2.mrc-lmb.cam.ac.uk/engineered-pol…

Behind the Paper | Nature Chemistry Community: chemistrycommunity.nature.com/posts/efficien…

(11/n)

Thank you to our co-workers, collaborators, and people who contributed to the story, i.a. @arangundy, Nithya Subramanian, Sew-Yeu Peak-Chew, @DNAmyWhitaker, @Freudlab, Misha Abramov, @PHerdewijn, @andimarx_lab & many more!

@MRC_LMB @Cambridge_Uni
#chembio #nucleicacids #synbio

@NatureChemistry