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Title: Synthesis and polymerase recognition of a pyrrolocytidine TNA triphosphate

Synthetic genetics is an area of synthetic biology that aims to extend the properties of heredity and evolution to artificial genetic polymers, commonly known as xeno‐nucleic acids or XNAs. In addition to establishing polymerases that are able to convert genetic information back and forth between DNA and XNA, efforts are underway to construct XNAs with expanded chemical functionality. α‐L‐Threose nucleic acid (TNA), a type of XNA that is recalcitrant to nuclease digestion and amenable to Darwinian evolution, provides a model system for developing XNAs with functional groups that are not present in natural DNA and RNA. Here, we describe the synthesis and polymerase activity of a cytidine TNA triphosphate analog (6‐phenyl‐pyrrolocytosine, tCpTP) that maintains Watson‐Crick base pairing with guanine. Polymerase‐mediated primer extension assays show that tCpTP is an efficient substrate for Kod‐RI, a DNA‐dependent TNA polymerase developed to explore the functional properties of TNA byin vitroselection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tCpTP and 7‐deaza‐tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available forin vitroselection.

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Wiley Blackwell (John Wiley & Sons)
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Medium: X
Sponsoring Org:
National Science Foundation
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