Systematic Analysis of 2'--Alkyl Modified Analogs for Enzymatic Synthesis and Their Oligonucleotide Properties

Overview

Journal Molecules

Publisher MDPI

Specialty Biology

Date 2023 Dec 9

PMID 38067640

Authors

Kenta Ishida

Yuuya Kasahara

Hidekazu Hoshino

Takumi Okuda

Satoshi Obika

Affiliations

Soon will be listed here.

Abstract

Enzymatic oligonucleotide synthesis is used for the development of functional oligonucleotides selected by in vitro selection. Expanding available sugar modifications for in vitro selection helps the functional oligonucleotides to be used as therapeutics reagents. We previously developed a KOD DNA polymerase mutant, KOD DGLNK, that enzymatically synthesized fully-LNA- or 2'--methyl-modified oligonucleotides. Here, we report a further expansion of the available 2'--alkyl-modified nucleotide for enzymatic synthesis by KOD DGLNK. We chemically synthesized five 2'--alkyl-5-methyluridine triphosphates and incorporated them into the oligonucleotides. We also enzymatically synthesized a 2'--alkyl-modified oligonucleotide with a random region (oligonucleotide libraries). The 2'--alkyl-modified oligonucleotide libraries showed high nuclease resistance and a wide range of hydrophobicity. Our synthesized 2'--alkyl-modified oligonucleotide libraries provide novel possibilities that can promote the development of functional molecules for therapeutic use.

References

Cozens C, Pinheiro V, Vaisman A, Woodgate R, Holliger P . A short adaptive path from DNA to RNA polymerases. Proc Natl Acad Sci U S A. 2012; 109(21):8067-72. PMC: 3361454. DOI: 10.1073/pnas.1120964109. View

Davies D, Gelinas A, Zhang C, Rohloff J, Carter J, OConnell D . Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets. Proc Natl Acad Sci U S A. 2012; 109(49):19971-6. PMC: 3523867. DOI: 10.1073/pnas.1213933109. View

Tuerk C, Gold L . Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990; 249(4968):505-10. DOI: 10.1126/science.2200121. View

Gold L, Ayers D, Bertino J, Bock C, Bock A, Brody E . Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS One. 2010; 5(12):e15004. PMC: 3000457. DOI: 10.1371/journal.pone.0015004. View

Ellington A, Szostak J . In vitro selection of RNA molecules that bind specific ligands. Nature. 1990; 346(6287):818-22. DOI: 10.1038/346818a0. View

Freier S, Altmann K . The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 1998; 25(22):4429-43. PMC: 147101. DOI: 10.1093/nar/25.22.4429. View

Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H . Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. J Biotechnol. 2001; 88(2):141-9. DOI: 10.1016/s0168-1656(01)00275-9. View

Jaffe G, Westby K, Csaky K, Mones J, Pearlman J, Patel S . C5 Inhibitor Avacincaptad Pegol for Geographic Atrophy Due to Age-Related Macular Degeneration: A Randomized Pivotal Phase 2/3 Trial. Ophthalmology. 2020; 128(4):576-586. DOI: 10.1016/j.ophtha.2020.08.027. View

Vaught J, Bock C, Carter J, Fitzwater T, Otis M, Schneider D . Expanding the chemistry of DNA for in vitro selection. J Am Chem Soc. 2010; 132(12):4141-51. DOI: 10.1021/ja908035g. View

10.

Dunn M, Otto C, Fenton K, Chaput J . Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures. ACS Chem Biol. 2016; 11(5):1210-9. DOI: 10.1021/acschembio.5b00949. View

11.

Masaki Y, Ito H, Oda Y, Yamazaki K, Tago N, Ohno K . Enzymatic synthesis and reverse transcription of RNAs incorporating 2'-O-carbamoyl uridine triphosphate. Chem Commun (Camb). 2016; 52(87):12889-12892. DOI: 10.1039/c6cc05796a. View

12.

Inoue H, Hayase Y, Imura A, Iwai S, Miura K, Ohtsuka E . Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides. Nucleic Acids Res. 1987; 15(15):6131-48. PMC: 306073. DOI: 10.1093/nar/15.15.6131. View

13.

Uemachi H, Kasahara Y, Tanaka K, Okuda T, Yoneda Y, Obika S . Hybrid-Type SELEX for the Selection of Artificial Nucleic Acid Aptamers Exhibiting Cell Internalization Activity. Pharmaceutics. 2021; 13(6). PMC: 8232673. DOI: 10.3390/pharmaceutics13060888. View

14.

Egli M, Manoharan M . Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 2023; 51(6):2529-2573. PMC: 10085713. DOI: 10.1093/nar/gkad067. View

15.

Chen T, Hongdilokkul N, Liu Z, Adhikary R, Tsuen S, Romesberg F . Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA. Nat Chem. 2016; 8(6):556-62. PMC: 4880425. DOI: 10.1038/nchem.2493. View

16.

Diafa S, Evequoz D, Leumann C, Hollenstein M . Enzymatic Synthesis of 7',5'-Bicyclo-DNA Oligonucleotides. Chem Asian J. 2017; 12(12):1347-1352. DOI: 10.1002/asia.201700374. View

17.

Gardner A, Jack W . Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases. Nucleic Acids Res. 2002; 30(2):605-13. PMC: 99817. DOI: 10.1093/nar/30.2.605. View

18.

Lesnik E, Guinosso C, Kawasaki A, Sasmor H, Zounes M, Cummins L . Oligodeoxynucleotides containing 2'-O-modified adenosine: synthesis and effects on stability of DNA:RNA duplexes. Biochemistry. 1993; 32(30):7832-8. DOI: 10.1021/bi00081a031. View

19.

Cummins L, Owens S, Risen L, Lesnik E, Freier S, McGee D . Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res. 1995; 23(11):2019-24. PMC: 306978. DOI: 10.1093/nar/23.11.2019. View

20.

Freund N, Taylor A, Arangundy-Franklin S, Subramanian N, Peak-Chew S, Whitaker A . A two-residue nascent-strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA. Nat Chem. 2022; 15(1):91-100. PMC: 7614059. DOI: 10.1038/s41557-022-01050-8. View