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DNA Polymerase Variants with High Processivity and Accuracy for Encoding and Decoding Locked Nucleic Acid Sequences.

Hidekazu Hoshino1,2, Yuuya Kasahara1,2, Masayasu Kuwahara3

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Researchers engineered KOD DNA polymerase variants to efficiently synthesize and transcribe locked nucleic acids (LNAs), a promising therapeutic molecule. These engineered polymerases enable the creation of novel LNA aptamers and catalysts, advancing nucleic acid therapeutics.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Xenobiotic nucleic acids (XNAs) are chemically modified analogues of DNA and RNA with therapeutic potential.
  • Locked nucleic acid (LNA) is a promising XNA, but its therapeutic development is hindered by a lack of efficient synthesis and reverse transcription tools.
  • Existing methods for LNA aptamer and catalyst development require specialized polymerases for DNA-to-LNA synthesis and LNA-to-DNA reverse transcription.

Purpose of the Study:

  • To develop engineered DNA polymerases capable of efficient LNA synthesis (encoding) and reverse transcription (decoding).
  • To enhance the performance of KOD DNA polymerase for creating LNA-based therapeutics.
  • To enable the synthesis of long LNA sequences and accommodate common therapeutic modifications like 2'-O-methyl (2'-OMe).

Main Methods:

  • Structural analysis of KOD DNA polymerase to guide protein engineering.
  • Site-directed mutagenesis to create LNA-decoding and encoding variants of KOD DNA polymerase.
  • Experimental validation of the engineered polymerases' efficiency, accuracy, and substrate acceptance, including synthesis of kilobase-length LNAs and SELEX for LNA aptamers.

Main Results:

  • Two KOD DNA polymerase variants, KOD DGLNK (DNA → LNA) and KOD DLK (LNA → DNA), were successfully engineered.
  • Both variants demonstrated significantly improved efficiency and accuracy in LNA synthesis and reverse transcription.
  • Kilobase-length LNAs were synthesized using KOD DGLNK, and the variants accepted 2'-O-methyl modifications, crucial for therapeutic applications.

Conclusions:

  • Engineered KOD DNA polymerase variants provide powerful tools for LNA synthesis and reverse transcription.
  • These variants overcome previous limitations, facilitating the development of novel LNA aptamers and catalysts.
  • The developed technology advances the field of XNA therapeutics by enabling the creation of molecules distinct from natural DNA and RNA.