Chiral-4'-Methyl-α-(l)-Threofuranosyl Nucleic Acids: Synthesis, Structure, Binding Affinity, Nuclease Resistance, and Polymerase Recognition.
- 1Alnylam Pharmaceuticals, 675 West Kendall Street, Cambridge, Massachusetts02142, United States.
- 2Department of Pharmaceutical Sciences, University of California, Irvine, California92697-3958, United States.
- 3Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee37232, United States.
Abstract
α-(l)-(3'-2')-Threofuranosyl nucleic acid (TNA) cross-pairs with DNA and RNA and is not degraded by nucleases and thus has potential for use in diagnostics and therapeutics based on nucleic acids. Here, we describe the synthesis of 15 building blocks of chiral 4'-(R)- and 15 building blocks of chiral (S)-methyl-α-(l)-threofuranosyl nucleic acid (4'-Me-TNA) monomers and their incorporation into oligonucleotides. Crystallographic studies of RNAs containing 4'-Me-TNA modifications revealed the impact of the methyl substituent on the sugar pucker and duplex conformation. Biostability studies showed that a terminal 4'-Me-TNA modification on a deoxyoligonucleotide imparted resistance to nuclease digestion equivalent to unmethylated TNA, which is markedly superior to that of more conventional 2'-ribo and 2'-deoxy modifications. A single nucleotide incorporation of the 4'-(R)-Me-TNA destabilized an otherwise all-RNA 12-mer duplex by 6.5 to 12 °C, depending on the base, relative to the unmodified RNA control duplex; the (S) isomer was less destabilizing than the (R) isomer. Computational models were used to rationalize the high levels of nuclease resistance and the thermal destabilization. The 4'-Me-TNA triphosphates are poorly recognized by an engineered TNA polymerase making it unlikely that the 4'-Me-TNA triphosphates will be recognized by natural polymerases. Together, these properties warrant the evaluation of 4'-Me-TNA in nucleic acid-based therapeutics.
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BibTeX
@article{mikami2026chiral4methylαlthreofuranosyl,
title = {{Chiral-4'-Methyl-α-(l)-Threofuranosyl Nucleic Acids: Synthesis, Structure, Binding Affinity, Nuclease Resistance, and Polymerase Recognition.}},
author = {Atsushi Mikami and Dhrubajyoti Datta and Jayanta Kundu and Vathan Kumar and Namrata Erande and Esau Medina and Victoria A Maola and Erica M Lee and Shigeo Matsuda and Joel M Harp and Martin Egli and John C Chaput and Muthiah Manoharan},
journal = {Journal of the American Chemical Society},
year = {2026},
month = sep,
doi = {10.1021/jacs.6c07051},
url = {https://europepmc.org/article/MED/42915539},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Chiral-4'-Methyl-α-(l)-Threofuranosyl Nucleic Acids: Synthesis, Structure, Binding Affinity, Nuclease Resistance, and Polymerase Recognition.
AU - Atsushi Mikami
AU - Dhrubajyoti Datta
AU - Jayanta Kundu
AU - Vathan Kumar
AU - Namrata Erande
AU - Esau Medina
AU - Victoria A Maola
AU - Erica M Lee
AU - Shigeo Matsuda
AU - Joel M Harp
AU - Martin Egli
AU - John C Chaput
AU - Muthiah Manoharan
PY - 2026
DA - 2026/09/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c07051
UR - https://europepmc.org/article/MED/42915539
AB - α-(l)-(3'-2')-Threofuranosyl nucleic acid (TNA) cross-pairs with DNA and RNA and is not degraded by nucleases and thus has potential for use in diagnostics and therapeutics based on nucleic acids. Here, we describe the synthesis of 15 building blocks of chiral 4'-(R)- and 15 building blocks of chiral (S)-methyl-α-(l)-threofuranosyl nucleic acid (4'-Me-TNA) monomers and their incorporation into oligonucleotides. Crystallographic studies of RNAs containing 4'-Me-TNA modifications revealed the impact of the methyl substituent on the sugar pucker and duplex conformation. Biostability studies showed that a terminal 4'-Me-TNA modification on a deoxyoligonucleotide imparted resistance to nuclease digestion equivalent to unmethylated TNA, which is markedly superior to that of more conventional 2'-ribo and 2'-deoxy modifications. A single nucleotide incorporation of the 4'-(R)-Me-TNA destabilized an otherwise all-RNA 12-mer duplex by 6.5 to 12 °C, depending on the base, relative to the unmodified RNA control duplex; the (S) isomer was less destabilizing than the (R) isomer. Computational models were used to rationalize the high levels of nuclease resistance and the thermal destabilization. The 4'-Me-TNA triphosphates are poorly recognized by an engineered TNA polymerase making it unlikely that the 4'-Me-TNA triphosphates will be recognized by natural polymerases. Together, these properties warrant the evaluation of 4'-Me-TNA in nucleic acid-based therapeutics.
SN - 0002-7863
SN - 1520-5126
ER -