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Published online 2 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 2 728-735
© 2004 Oxford University Press

PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1

A. Michael Sismour, Stefan Lutz1, Jeong-Ho Park2, Michael J. Lutz3, Paul L. Boyer4, Stephen H. Hughes4 and Steven A. Benner*

Departments of Chemistry and Anatomy and Cell Biology, University of Florida, Gainesville, FL 32611-7200, USA, 1 Department of Chemistry, Emory University, Atlanta, GA 30322, USA, 2 Department of Chemical Technology, Hanbat National University, Taejon 305-719, South Korea, 3 Novartis Pharma, Basel CH-4002 Switzerland and 4 HIV Drug Resistance Program, National Cancer Institute, NCI-Frederick, Frederick, MD 21702-1201, USA

*To whom correspondence should be addressed. Tel: +1 352 392 7773; Fax: +1 352 392 7918; Email: benner{at}chem.ufl.edu

As the next step towards generating a synthetic biology from artificial genetic information systems, we have examined variants of HIV reverse transcriptase (RT) for their ability to synthesize duplex DNA incorporating the non-standard base pair between 2,4-diaminopyrimidine (pyDAD), a pyrimidine presenting a hydrogen bond ‘donor–acceptor–donor’ pattern to the complementary base, and xanthine (puADA), a purine presenting a hydrogen bond ‘acceptor–donor–acceptor’ pattern. This base pair fits the Watson–Crick geometry, but is joined by a pattern of hydrogen bond donor and acceptor groups different from those joining the GC and AT pairs. A variant of HIV-RT where Tyr 188 is replaced by Leu, has emerged from experiments where HIV was challenged to grow in the presence of drugs targeted against the RT, such as L-697639, TIBO and nevirapine. These drugs bind at a site near, but not in, the active site. This variant accepts the pyDAD-puADA base pair significantly better than wild type HIV-RT, and we used this as a starting point. A second mutation, E478Q, was introduced into the Y188L variant, in the event that the residual nuclease activity observed is due to the RT, and not a contaminant. The doubly mutated RT incorporated the non-standard pair with sufficient fidelity that the variant could be used to amplify oligonucleotides containing pyDAD and puADA through several rounds of a polymerase chain reaction (PCR) without losing the non-standard base pair. This is the first time where DNA containing non-standard base pairs with alternative hydrogen bonding patterns has been amplified by a full PCR. This work also illustrates a research strategy that combines in clinico pre-evolution of proteins followed by rational design to obtain an enzyme that meets a particular technological specification.


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