Oligothymidylate analogs having stereoregular, alternating methylphosphonate/phosphodiester backbones as primers for DNA polymerase

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Abstract
Oligothymidylate analogs having stereoregular, alternating methylphosphonate[p]/phosphodiester [p] backbones, d-Tp(TpTp)4T isomers I and II and d-Tp(TpTp)3T(pT)1-5 isomers I and II, were prepared by methods analogous to the phosphotriester synthetic technique. The designations isomer I and isomer II refer to the configuration of the methylphosphonate linkage, which is the same throughout each isomer. Analogs with the type I methylphosphonate configuration from very stable duplexes with poly(dA) while those with the type II configuration from either 2T:1A triplexes or 1T:1A duplexes with poly(dA) of considerably lower stabilities. The oligothymidylate analogs were tested for their ability to initiate polymerizations catalyzed by Escherichia coli DNA polymerase I or calf thymus DNA polymerase .alpha. on a poly(dA) template. Neither d-Tp(TpTp)4T nor d-Tp(TpTp)3TpT served as initiators of polymerization while d-Tp(TpTp)3T(pT)2-5 showed increasing priming ability as the length of the 3''-oligothymidylate tail increased. Analogs with type I methylphosphonate configuration were more effective initiators than the type II analogs at 37.degree. C. The apparent activation energies of polymerizations initiated by d-Tp(TpTp)3T-(pT)4 and 5 isomer I were greater than those for reactions initiated by isomer II or d-(Tp)11T. DNA polymerase probably interacts with the charged phosphodiester groups of the primer molecule and may help stabilize primer/template interaction. At least 2 contiguous phosphodiester groups are required at the 3'' end of the analog primers in order to for polymerization to occur. Interactions between the polymerase and primer also appear to occur with phosphodiester groups located at sites remote from the 3''-OH polymerization site and may be influenced by the configuration of the methylphosphonate group.