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Overcoming exercise limitations of oligonucleotide catalysts by way of artificial genetics


The capability of single-stranded nucleic acids to kind not simply duplexes with a complementary strand, however to fold into elaborate tertiary buildings underpins the combinatorial energy of nucleic acids: from deceptively easy chemical constructing blocks emerge complicated buildings with subtle biochemical features.

Amongst these, RNA molecules with catalytic exercise should not solely on the coronary heart of basic processes in fashionable biology – together with protein synthesis and mRNA and tRNA processing – however are broadly believed to have been instrumental on the origin of life. The invention of such RNA enzymes (ribozymes), honoured with the award of the 1989 Nobel prize1, supplied the primary impetus for a deeper examination of the catalytic potential of pure nucleic acids in addition to artificial analogues.

Small trans-acting oligonucleotide catalysts able to particular RNA cleavage – such because the hammerhead ribozyme, and laboratory-evolved analogous DNAzymes 10-23 and 8-172,3 – particularly have been intensively studied by artificial chemical biologists for many years due to their nice potential for medical and biotechnological functions. Nevertheless, though some variations of these molecules are exhibiting promise in diagnostic functions, the profitable utility of such catalysts as programmable, chemically-synthesised therapeutic brokers for precision knockdown of disease-associated RNA transcripts has been hindered by the constraints inherent in pure nucleic acid chemistry. Though scientific trials have repeatedly demonstrated the security and tolerability of ribozymes and DNAzymes in vivo, susceptibility to nuclease degradation, dependency on unphysiological focus of steel ion cofactors, poor substrate accessibility inside structured goal RNAs and poor intracellular supply have remained formidable obstacles to attaining scientific affect.

Systematic exploitation of insights gained from > 40 years of nucleic acid analogue chemistry has confirmed essential for the interpretation of practical nucleic acid applied sciences to the clinic (antisense oligonucleotides (ASOs), silencing RNA (siRNA, RNAi) and artificial mRNA)4.  Nevertheless, significantly much less effort had gone into exploring modified nucleic acid chemistries within the context of ribozymes and DNAzymes with a view to addressing their limitations. Unequivocal demonstrations of particular cleavage exercise, not simply on quick mannequin substrates utilizing excessive concentrations (≥10 mM) of divalent steel ions (usually Mg2+), however in specific on lengthy, structured RNAs and full mRNA transcripts in physiologically related situations (≤1 mM [Mg2+]) has remained elusive.

Whereas in vivo (i.e. intracellular) RNA knockdown results have been reported following DNAzyme transfections, such information have to be interpreted with warning because of the variety of pitfalls and sources of false-positives that exist when measuring such results, as we define in a current Issues Arising article5. Certainly, results in cells have been reported even when DNAzyme catalytic exercise has been inactivated6 (as additionally noticed with inactive ribozymes 30 years in the past7), suggesting that they’ve the capability to behave as antisense oligonucleotides in vivo, lowering the extent of goal RNAs by way of easy base-pairing and recruitment of host silencing equipment (mainly RNase H1) relatively than intrinsic enzymatic RNA chopping. 

The excellence between an ASO-like motion and DNAzyme-mediated cleavage is essential, as with out it the principal benefits of DNAzyme know-how – excessive cleavage specificity and host factor-independence – are misplaced.

Our curiosity in this space was sparked following our 2012 discovery that fully-modified synthetic analogues of DNA and RNA not present in nature – referred to as xeno nucleic acids (XNA) – couldn’t solely function artificial genetic polymers8 however may very well be developed into practical buildings (initially, XNA ligands or aptamers)9. We postulated that the chemical construction area of any information-carrying polymer able to positioning practical teams in 3D might, in precept, help the evolution of enzymes and in 2015 we have been capable of verify this by way of the directed evolution of a vary of catalysts fully composed of a wide range of XNA chemistries, “XNAzymes”, with RNA-endonuclease and RNA- and XNA-ligase actions10,11.

We reasoned that the aptitude of XNA chemistries equivalent to 2’-deoxy-2’-fluoro-β-D-arabino nucleic acid (FANA) to kind buildings with enhanced stability in contrast with DNA or RNA would possibly alleviate the requirement for divalent steel ions for folding and catalysis. Regardless of this, the primary technology of RNA-cleaving XNAzymes exhibited poor exercise on all-RNA substrates in physiological magnesium concentrations (0.5 – 1 mM Mg2+) nor might they be simply reprogrammed to cleave different goal sequences; exercise shortly dropped off as substrate binding arms have been cumulatively mutated. Nevertheless, after modifying our directed evolution workflow and choice methods for the discovery of XNAzymes able to cleaving extra ‘practical’ goal RNAs underneath low [Mg2+], we have been capable of determine a brand new all-FANA catalytic motif  FR6_1 – that not solely outperformed earlier XNAzymes, however retained its RNA cleavage exercise in physiological situations. Moreover, FR6_1 may very well be readily re-targeted to cleave a variety of extremely structured full-length (>5 kb) (m)RNA targets.

By exploiting the FR6_1 XNAzyme’s excessive RNA cleavage specificity, in addition to the ‘programmability’ of substrate-binding arm sequences, we have been capable of exhibit a key proof-of-concept of the potential energy of the know-how: cleaving practical RNA transcripts or substrates with single-nucleotide precision, on this case cancer-associated mutants of traditional oncogenes KRAS [G12D] (Fig. 1) and BRAF [V600E]. This enabled allele-specific mRNA knockdown of G12D (c.G35A) KRAS mRNA inside hours of transfection into reside cultured adenocarcinoma cells, each in a KRAS G12D homozygous and heterozygous genetic background, within the presence of wild-type (c.G35) mRNA.

Determine 1. An XNAzyme able to allele-specific cleavage of KRAS [G12D] mRNA.
A variant of the FR6_1 FANA XNAzyme focused to codon 12 within the mRNA of the oncogene KRAS, a cancer-associated mutation hotspot. XNAzyme “FR6_1_KRas12B” particularly cleaves the full-length transcript encoding the frequent mutation c.35G>A [G12D], however not the wild sort, in physiological situations in vitro and inside reside colon carcinoma cells. Measured by droplet digital RT-qPCR.

Though we discover that FANA XNAzymes proceed to induce a (much less particular) antisense-mediated knockdown, we are able to now disentangle these contributions from (allele-specific) XNAzyme catalysis by growth and validation of stringent controls, a catalytically-inactive level mutant of FR6_1 and an analogous variant of the traditional 10-23 DNAzyme. This additionally concerned substantial efforts to eradicate technical artefacts related to generally used methods for mobile RNA workup and measurement of gene silencing by qRT-PCR that aren’t ideally suited to work with XNAzymes or XNA-modified DNAzymes. On account of their increased base-pairing stability and partial resistance to nuclease degradation these can stay lively in mobile extracts throughout DNase therapy and subsequent RT steps, yielding false optimistic knockdown. Subsequently, nice care have to be taken to take away them from mRNA preparations to guarantee measurements really replicate intracellular exercise. Utilizing these stringent controls, we exhibit that roughly 50% of FR6_1 XNAzyme-mediated knockdown happens on account of the bona fide catalytic turnover of the XNAzyme inside cells, distinct from antisense-type results. 

Our outcomes underline the potential of XNA know-how to beat a few of the long-standing difficulties related with oligonucleotide catalysts, particularly in enabling substantial practical beneficial properties underneath physiological situations and towards practical targets, offering a basis for the extra systematic growth of improved gene silencing brokers based mostly on FANA or different XNA chemistries. Certainly, chemistries much more sturdy than FANA, equivalent to 2’OMe-, 2’-MOE-RNA or locked nucleic acids (LNA), with considerably diminished or abolished capability to set off RNase H, provide promise for the discovery of next-generation XNAzymes with ever extra exact and long-lasting in vivo actions. Though technically difficult, incorporation of expanded nucleobases in addition to spine chemistry into XNAzyme choices additionally suggests a path to catalysts with a larger variety of response mechanisms. Bringing mechanisms analogous to these employed by proteinaceous enzymes into the realm of oligo catalysts would offer substantial beneficial properties in catalytic turnover12 enabling improved knockdown and decrease efficient doses – in addition to the thrilling prospect of catalysts for reactions and chemical environments inaccessible to enzymes composed of pure polymers.

References:

1          The Nobel Prize in Chemistry 1989. https://www.nobelprize.org/prizes/chemistry/1989/press-release/

2          Faulhammer, D. & Famulok, M. Characterization and divalent metal-ion dependence of in vitro chosen deoxyribozymes which cleave DNA/RNA chimeric oligonucleotides. Journal of Molecular Biology 269, 188-202 (1997). https://doi.org/10.1006/jmbi.1997.1036 

3          Santoro, S. W. & Joyce, G. F. A basic goal RNA-cleaving DNA enzyme. Proceedings of the Nationwide Academy of Sciences of the US of America 94, 4262-4266 (1997). https://doi.org/10.1073/pnas.94.9.4262

4          Gait, M. J. & Agrawal, S. Introduction and historical past of the chemistry of nucleic acids therapeutics. Strategies in Molecular Biology 2434, 3-31 (2022). https://doi.org/10.1007/978-1-0716-2010-6_1

5          Taylor, A. I. & Holliger, P. On gene silencing by the X10-23 DNAzyme. Nature Chemistry (2022). https://doi.org/10.1038/s41557-022-00990-5

6          Younger, D. D., Full of life, M. O. & Dieters, A. Activation and deactivation of DNAzyme and antisense operate with mild for the photochemical regulation of gene expression in mammalian cells. Journal of the American Chemical Society 132, 6183-6193 (2010). https://doi.org/10.1021/ja100710j 

7          Steinecke, P., Herget, T. & Schreier, P. H. Expression of a chimeric ribozyme gene leads to endonucleolytic cleavage of goal mRNA and a concomitant discount of gene expression in vivo. The EMBO Journal 11, 1525-1530 (1992). https://doi.org/10.1002/j.1460-2075.1992.tb05197.x  

8          Pinheiro, V. B., Taylor, A. I., Cozens, C., Abramov, M., Renders, M., Zhang, S., Chaput, J. C., Wengel, J., Peak-Chew, S.-Y., McLaughlin, S. H., Herdewijn, P. & Holliger, P. Artificial genetic polymers able to heredity and evolution. Science 336, 341-344 (2012). https://doi.org/10.1126/science.1217622

9          Taylor, A. I., Houlihan, G. & Holliger, P. Past DNA and RNA: The increasing toolbox of artificial genetics. Chilly Spring Harbor Views in Biology 11, a032490 (2019). https://doi.org/10.1101/cshperspect.a032490

10          Taylor, A. I. & Holliger, P. Directed evolution of synthetic enzymes (XNAzymes) from various repertoires of artificial genetic polymers. Nature Protocols 10, 1625-1642 (2015). https://doi.org/10.1038/nprot.2015.104

11        Taylor, A. I., Pinheiro, V. B., Smola, M. J., Morgunov, A. S., Peak-Chew, S.-Y., Cozens, C., Weeks, Okay. M., Herdewijn, P. & Holliger, P. Catalysts from artificial genetic polymers. Nature 518, 427-430 (2015). https://doi.org/10.1038/nature13982

12        Wang, Y., Liu, E., Lam, C. H. & Perrin, D. M. A densely modified M2+-independent DNAzyme that cleaves RNA effectively with a number of catalytic turnover. Chemical Science 9, 1813-1821 (2018). https://doi.org/10.1039/C7SC04491G

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