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Aaron M Leconte

Publications and source records attributed to Aaron M Leconte.

6 recordsLinked to original sources
PubMed2006

An efficiently extended class of unnatural base pairs.

A third DNA base pair, which is synthesized efficiently and selectively, would have wide ranging applications from synthetic organisms to nucleic acids biotechnology. Hydrophobic unnatural nucleobases offer a promising route to such a pair, but are often limited by inefficient extension, defined as synthesis immediately following the unnatural pair. Here, we describe a simple screen which enables the characterization of large numbers of previously uncharacterized hetero base pairs. From this screen, we identified a class of unnatural base pairs which are extended more efficiently than any unnatural base pair reported to date. Screening, when complemented by further kinetic analysis, can improve the understanding of the determinants of efficient extension as well as identify viable hetero base pairs.

Base Pairing↗
PubMed2005

Polymerase evolution: efforts toward expansion of the genetic code.

Genetic information is encoded by, but potentially not limited to, a four-letter alphabet. A variety of predominantly hydrophobic nucleobase analogues that form self-pairs in DNA have been examined as third base pair candidates. For example, the PICS self-pair is both stable in duplex DNA and synthesized by some wild-type polymerases with reasonable efficiency. These efforts to expand the genetic code are expected to be facilitated by optimizing both the unnatural nucleobase analogues and the polymerases that replicate them. Here, we report the use of an activity-based selection system to evolve a DNA polymerase that more efficiently replicates DNA containing the PICS self-pair. The selection system is based on the co-display on phage of DNA polymerase libraries and a DNA substrate containing the self-pair. Only polymerases that accept the unnatural substrate incorporate a biotin-dUTP to the attached primer and may then be isolated on a streptavidin solid support. A mutant of Sf polymerase, P2, was evolved which both inserts dPICSTP opposite dPICS in the template and extends the unnatural primer terminus by incorporation of the next correct natural dNTP, where the parental enzyme catalyzes neither step at detectable rates. P2 was found to be a triple mutant of Sf, with the mutations F598I, I614F, and Q489H. The evolved properties of P2, as well as the observed mutations, are consistent with an increased affinity for the DNA primer-template containing the self-pair.

DNA↗