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Evolving a Polymerase for Hydrophobic Base Analogues

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MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom, and Department of Chemistry, Stanford University, Stanford, California 94305
†MRC Laboratory of Molecular Biology.
‡Present address: Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, 46012 Valencia, Spain.
§Stanford University.
Cite this: J. Am. Chem. Soc. 2009, 131, 41, 14827–14837
Publication Date (Web):September 24, 2009
Copyright © 2009 American Chemical Society

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    Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson−Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: ϕ (abasic site), d5NI: ϕ, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

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    Protein sequences of selected polymerases (including 5D4), additional data on kinetic constants, substrate specificity, primer extension, NMR spectroscopy, reversion mutant activity, SCA and FoldX analysis. This material is available free of charge via the Internet at

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