title: Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs.
abstract: Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d5SICS and analogues of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new dMMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, dNaM-d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure-activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogues with the exception of dNaM. We also found that dNaM-d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lavergne
authors.forename: Denis A
authors.initials: DA
authors.lastname: Malyshev
authors.forename: Floyd E
authors.initials: FE
authors.lastname: Romesberg
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D015394:Molecular Structure
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase