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Mutant polymerases capable of 2' fluoro-modified nucleic acid synthesis and amplification with improved accuracy.

ABSTRACT: Nonnatural nucleic acids (xeno nucleic acids, XNA) can possess several useful properties such as expanded reactivity and nuclease resistance, which can enhance the utility of DNA as a biotechnological tool. Native DNA polymerases are unable to synthesize XNA, so, in recent years mutant XNA polymerases have been engineered with sufficient activity for use in processes such as PCR. While substantial improvements have been made, accuracy still needs to be increased by orders of magnitude to approach natural error rates and make XNA polymerases useful for applications that require high fidelity. Here, we systematically evaluate leading Taq DNA polymerase mutants for their fidelity during synthesis of 2'F XNA. To further improve their accuracy, we add mutations that have been shown to increase the fidelity of wild-type Taq polymerases, to some of the best current XNA polymerases (SFM4-3, SFM4-6, and SFP1). The resulting polymerases show significant improvements in synthesis accuracy. In addition to generating more accurate XNA polymerases, this study also informs future polymerase engineering efforts by demonstrating that mutations that improve the accuracy of DNA synthesis may also have utility in improving the accuracy of XNA synthesis.

SUBMITTER: Christensen TA

PROVIDER: S-EPMC9347352 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Mutant polymerases capable of 2' fluoro-modified nucleic acid synthesis and amplification with improved accuracy.

Christensen Trevor A TA Lee Kristi Y KY Gottlieb Simone Z P SZP Carrier Mikayla B MB Leconte Aaron M AM

RSC chemical biology 20220617 8

Nonnatural nucleic acids (xeno nucleic acids, XNA) can possess several useful properties such as expanded reactivity and nuclease resistance, which can enhance the utility of DNA as a biotechnological tool. Native DNA polymerases are unable to synthesize XNA, so, in recent years mutant XNA polymerases have been engineered with sufficient activity for use in processes such as PCR. While substantial improvements have been made, accuracy still needs to be increased by orders of magnitude to approac ...[more]

PMID: 35975008

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Project description:We report the design and synthesis of 2'-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-D-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2'-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3'-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2'-fluoro RNA (FRNA), 2'-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the (3)H(2) and (2)H(3) conformations, similar to the C3'-endo/C2'-endo conformation equilibrium seen in natural furanose nucleosides. In the (2)H(3) conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3'-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the (3)H(2) (C3'-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d(GCGTA)-T*-d(ACGC)](2.) In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications.

Dataset Information

Mutant polymerases capable of 2' fluoro-modified nucleic acid synthesis and amplification with improved accuracy.

Publications

Mutant polymerases capable of 2' fluoro-modified nucleic acid synthesis and amplification with improved accuracy.

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