Major Groove Substituents and Polymerase Recognition of a Class of Predominantly Hydrophobic Unnatural Base Pairs

摘要

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 d>5SICS and analogues of d>MMO2, 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 d>MMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d>5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, d>NaM-d>5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases is 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, d>NMO1, that is a better partner for d>5SICS than any of the previously identified d>MMO2 analogues with the exception of d>NaM. We also find that d>NaM-d>5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.