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

Abstract:

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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