In vitro evolution of ribonucleases from expanded genetic alphabets

Abstract

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucleotide building blocks is limited when compared with that of proteins. Here, we assess the evolutionary potential of libraries of nucleic acids with six nucleotide building blocks as reservoirs for catalysis. We compare the outcomes of in vitro selection experiments toward RNA-cleavage activity of two nucleic acid libraries: one built from the standard four independently replicable nucleotides and the other from six, with the two added nucleotides coming from an artificially expanded genetic information system (AEGIS). Results from comparative experiments suggest that DNA libraries with increased chemical diversity, higher information density, and larger searchable sequence spaces are one order of magnitude richer reservoirs of molecules that catalyze the cleavage of a phosphodiester bond in RNA than DNA libraries built from a standard four-nucleotide alphabet. Evolved AEGISzymes with nitro-carrying nucleobase Z appear to exploit a general acid–base catalytic mechanism to cleave that bond, analogous to the mechanism of the ribonuclease A family of protein enzymes and heavily modified DNAzymes. The AEGISzyme described here represents a new type of catalysts evolved from libraries built from expanded genetic alphabets.

How proton transfer impacts hachimoji DNA

“…Understanding the intrinsic stability of hachimoji DNA and the relative populations of their zwitterionic and tautomeric forms could impact medicinal chemistry and the development of antisense therapeutics and articial oligo chemistries. [70][71][72][73][74][75] Lastly, we highlight some limitations of the present study and the need for future work to determine the minimum free energy pathway of the proton transfer schemes explored in this paper. Several QM/MM studies on proton transfer in DNA highlight that complex interactions with the solvent, the rest of the DNA structure, and the DNA replisome could signicantly alter the DNA shape.…”

“…Understanding the intrinsic stability of hachimoji DNA and the relative populations of their zwitterionic and tautomeric forms could impact medicinal chemistry and the development of antisense therapeutics and artificial oligo chemistries. 70–75…”

Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores

“…These are exactly the kinds of sequences generated by laboratory in vitro evolution (LIVE) applied to hachimoji libraries. Here, the binding specificity and enzyme catalytic activity of aptamers and aptazymes can be greatly increased compared to standard NAs through the addition of only a few nonstandard hachimoji nucleotides. ,− , This is one of many current and near-future applications of hachimoji NAs with sparse incorporation of nonstandard hachimoji nucleotides.…”

“…24 This has, for example, enabled deep sequencing 6letter GACTZP DNA molecules that have been evolved to bind cancer cells, 25 block the action of toxins, 11 and cleave RNA with sequence specificity. 26 Other conceivable sequencing approaches might include mass spectrometry 7 and sequencing by hybridization. 27 Nanopore sequencing 28 is an attractive option for sequencing nonstandard DNA.…”

Rethinking nucleic acids from their origins to their applications

“…The advantages of AEGIS-LIVE over standard LIVE is exceptionally evident when catalysts are sought. In another set of experiments, Elisa Biondi did parallel in vitro selection experiments to evolve RNA-cleaving DNAzymes with libraries containing either standard DNA, or AEGIS DNA built from six nucleotides (ACTG ZP ) [110]. The starting libraries contained 25 nucleotides in the random regions composed of either ACTG, or ACTG ZP nucleotides, surrounded by standard DNA primer binding sites.…”