title: 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.
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA.
authors.forename: A Michael
authors.initials: AM
authors.lastname: Sismour
authors.forename: Stefan
authors.initials: S
authors.lastname: Lutz
authors.forename: Jeong-Ho
authors.initials: JH
authors.lastname: Park
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lutz
authors.forename: Paul L
authors.initials: PL
authors.lastname: Boyer
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Hughes
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D020029:Base Pairing; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D014644:Genetic Variation / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D004247:DNA; C088944:Deep Vent DNA polymerase; D019914:Taq Polymerase; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase