Detection of a guanine X adenine base pair in a decadeoxyribonucleotide by proton magnetic resonance spectroscopy.

A decadeoxyribonucleotide, d(C-C-A-A-G-A-T-T-G-G) (I), forms a duplex in solution. The base pairing pattern in this duplex was studied by proton nuclear magnetic resonance spectroscopy. Five NH...N hydrogen-bonded proton resonances were observed, and they were assigned by nuclear Overhauser enhancement experiments as well as by comparison to five previously assigned NH...N hydrogen-bonded proton resonances in a self-complementary duplex of similar sequence, d(C-C-A-A-G-C-T-T-G-G) (II). The results suggest that the central -G-A- residues of I form G X A base pairs in the helical state. The fact that the H2 proton of A at the sixth position from the 5' end of I showed nuclear Overhauser enhancement when the NH...N hydrogen-bonded proton resonance of G X A was irradiated suggests that the bases of the G X A base pair are oriented in an anti-anti conformation. Comparison of the linewidths at the half height of the NH...N hydrogen-bonded proton resonances of I at 1 degree C suggest that the G X A base pairs are less stable than adjacent A X T base pairs.

Synthesis and template properties of an ethyl phosphotriester modified decadeoxyribonucleotide.

The phosphate groups of nucleic acids are often the targets of mutagenic and carcinogenic alkylating agents. In order to study the effects of alkyl phosphotriester modification on the physical and biochemical properties of DNA, two diastereomeric ethyl phosphotriester modified decadeoxyribonucleotides, d-CpCpApApGp(Et)ApTpTpGpG isomer I and isomer II, were prepared. A phosphotriester synthetic procedure was used to specifically place ethyl triester groups with either an R or S configuration in the central dimer region of the decamer. Terminal deoxynucleotidyl transferase was used to add oligodeoxyadenylate tails to the 3' end of the decamers. The resulting oligomers were tested as templates for Escherichia coli DNA polymerase I with d-(pT)8pCpC as a primer. The rates and extents of polymerization directed by the modified templates were 25% (isomer I) and 50% (isomer II) less than those of an unmodified control template. Thus the presence of an ethyl triester group inhibits polymerization, the effectiveness of which is determined by the orientation of the ethyl group relative to the rest of the template backbone. These results suggest ethyl phosphotriester lesions could inhibit replication rates of cellular DNA.

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

Oligothymidylate analogues having stereoregular, alternating methylphosphonate/phosphodiester 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 nd isomer II refer to the configuration of the methylphosphonate linkage, which is the same through each isomer. Analogues with the type I methylphosphonate configuration form very stable duplexes with poly(dA) while those with the type II configuration form either 2T:1A triplexes or 1T:1A duplexes with poly(dA) of considerably lower stabilities. The oligothymidylate analogues 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(T]Tp)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. Analogues with type I methylphosphonate configuration were more effective initiators than the type II analogues at 37 degrees 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. The results suggest that DNA polymerase interacts with the charged phosphodiester groups of the primer molecule and may help stabilize primer/template interaction. At least two contiguous phosphodiester groups are required at the 3' end of the analogue primers in order 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.

Oligothymidylate analogues having stereoregular, alternating methylphosphonate/phosphodiester backbones. Synthesis and physical studies.

Two decathymidylate analogues, d-(TpTp)4TpT-isomer 1 and isomer 2, having stereoregular, alternating methylphosphonate/phosphodiester backbones were prepared. The phosphodiester linkages of d-(TpTp)4TpT are cleaved slowly by snake venom phosphodiesterase in a stepwise manner, while slow random cleavage occurs with micrococcal nuclease which hydrolyzes isomer 2 faster than isomer 1. The CD spectra of isomer 1 and d-(Tp)9T are identical suggesting they have similar conformations, while that of isomer 2 shows an overall reduction of [theta]. Isomer 1 forms a 1T . 1A complex with poly(dA) and both 1T . 1A and 2T . 1A complexes with poly(rA), while isomer 2 forms a 2T . 1A complex of low thermal stability with poly(dA) and no complex with poly(rA). The Tm values of the partially nonionic d-(TpTp)4TpT . polynucleotide complexes are less dependent on salt concentration than are those of d-(Tp)9T. The stoichiometry and CD spectra of the complexes suggest that poly(dA) . isomer 1 duplex assumes a B-type geometry while isomer 2 . poly(dA) . isomer 2 triplex and the isomer 1 . poly(rA) complexes have an A-type geometry. Although there are no apparent differences between steric restrictions to rotation about the backbones of either isomer 1 or 2, or steric restrictions to complex formation, the results suggest that the configuration of the methylphosphate linkage controls: 1) interaction with nucleases, 2) oligomer conformation, and 3) interaction with polynucleotides. The latter effects may result from differences in solvation of the two isomers.

Preparation of a decadeoxyribonucleotide helix for studies by nuclear magnetic resonance.

A self-complementary decadeoxyribonucleotide d-CpCpApApGpCpTpTpGpG was chemically synthesized by a procedure based on the phosphotriester approach. This procedure was carefully monitored and appropriately modified to ensure the purity of oligomer components at each step of the synthetic scheme. Extensive use was made of both analytical and preparative high-pressure liquid chromatography to purify and characterize the decamer and its constituent oligonucleotides. The final product (1318 A257 units or 16.5 mumol) was obtained in high purity and sufficient quantity for extensive physical studies by UV, CD, and NMR spectroscopy. Our preliminary results show that at a strand concentration of 1.3 X 10(-5) M and in 0.10 M sodium chloride and 0.01 M sodium phosphate buffer, pH 7.0, the decamer duplex has a Tm at 47 degrees C. The CD spectrum of this decamer duplex is similar to that of B-form DNA. All the resonances of the nonexchangeable base protons of the decamer are well resolved in the 1H NMR spectrum, when the single-stranded form was examined by using a 360-MHz spectrometer and when the duplex form was examined by using a 600-MHz spectrometer. These base proton resonances have been tentatively assigned by using the incremental assignment technique. Although the decamer duplex serves as a substrate for AluI restriction endonuclease, it is not cleaved by HindIII endonuclease.