Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′-5′ Linkages

Christopher Cozens; Hannes Mutschler; Geoffrey M. Nelson; Gillian Houlihan; Alexander I. Taylor; Philipp Holliger

doi:10.1002/anie.201508678

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′-5′ Linkages

Christopher Cozens, Hannes Mutschler, Geoffrey M. Nelson, Gillian Houlihan, Alexander I. Taylor, Philipp Holliger^*

^*Corresponding author for this work

Chemistry

University of Cambridge

Research output: Contribution to journal › Article › peer-review

22 Citations (Scopus)

Abstract

Information-bearing nucleic acids display universal 3′-5′ linkages, but regioisomeric 2′-5′ linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2′-5′ linkages by an engineered polymerase using 3′-deoxy- or 3′-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3′-5′ linked DNA with good fidelity. This enables a fast and simple method for "structural mutagenesis" by the position-selective incorporation of 2′-5′ linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23RNA endonuclease DNAzyme.

Original language	English
Pages (from-to)	15570-15573
Number of pages	4
Journal	Angewandte Chemie - International Edition
Volume	54
Issue number	51
DOIs	https://doi.org/10.1002/anie.201508678
Publication status	Published - 14 Dec 2015

Keywords

DNAzyme
nucleic acids
nucleotides
polymerase
regioselectivity

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10.1002/anie.201508678

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title = "Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′-5′ Linkages",

abstract = "Information-bearing nucleic acids display universal 3′-5′ linkages, but regioisomeric 2′-5′ linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2′-5′ linkages by an engineered polymerase using 3′-deoxy- or 3′-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3′-5′ linked DNA with good fidelity. This enables a fast and simple method for {"}structural mutagenesis{"} by the position-selective incorporation of 2′-5′ linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23RNA endonuclease DNAzyme.",

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AU - Cozens, Christopher

AU - Mutschler, Hannes

AU - Nelson, Geoffrey M.

AU - Houlihan, Gillian

AU - Taylor, Alexander I.

AU - Holliger, Philipp

PY - 2015/12/14

Y1 - 2015/12/14

N2 - Information-bearing nucleic acids display universal 3′-5′ linkages, but regioisomeric 2′-5′ linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2′-5′ linkages by an engineered polymerase using 3′-deoxy- or 3′-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3′-5′ linked DNA with good fidelity. This enables a fast and simple method for "structural mutagenesis" by the position-selective incorporation of 2′-5′ linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23RNA endonuclease DNAzyme.

AB - Information-bearing nucleic acids display universal 3′-5′ linkages, but regioisomeric 2′-5′ linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2′-5′ linkages by an engineered polymerase using 3′-deoxy- or 3′-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3′-5′ linked DNA with good fidelity. This enables a fast and simple method for "structural mutagenesis" by the position-selective incorporation of 2′-5′ linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23RNA endonuclease DNAzyme.

KW - DNAzyme

KW - nucleic acids

KW - nucleotides

KW - polymerase

KW - regioselectivity

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ER -

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′-5′ Linkages

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Keywords

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