Conformational properties of poly[d(G-T)].poly[d(C-A)] and poly[d(A-T)] in low- and high-salt solutions: NMR and laser Raman analysis.

Abstract

Abstract31P‐ and 1H‐nmr and laser Raman spectra have been obtained for poly[d(G‐T)]·[d(C‐A)] and poly[d(A‐T)] as a function of both temperature and salt. The 31P spectrum of poly[d(G‐T)]·[d(C‐A)] appears as a quadruplet whose resonances undergo separation upon addition of CsCl to 5.5M. 1H‐nmr measurements are assigned and reported as a function of temperature and CsCl concentration. One dimensional nuclear Overhauser effect (NOE) difference spectra are also reported for poly[d(G‐T)]·[d(C‐A)] at low salt. NOE enhancements between the H8 protons of the purines and the C5 protons of the pyrimidines, (H and CH3) and between the base and H‐2′,2″ protons indicate a right‐handed B‐DNA conformation for this polymer. The NOE patterns for the TH3 and GH1 protons in H2O indicate a Watson–Crick hydrogen‐bonding scheme. At high CsCl concentrations there are upfield shifts for selected sugar protons and the AH2 proton. In addition, laser Raman spectra for poly[d(A‐T)] and poly[d(G‐T)]·[d(C‐A)] indicate B‐type conformations in low and high CsCl, with predominantly C2′‐endo sugar conformations for both polymers. Also, changes in base‐ring vibrations indicate that Cs+ binds to O2 of thymine and possibly N3 of adenine in poly[d(G‐T)]·[d(C‐A)] but not in poly[d(A‐T)]. Further, 1H measurements are reported for poly[d(A‐T)] as a function of temperature in high CsCl concentrations. On going to high CsCl there are selective upfield shifts, with the most dramatic being observed for TH1′. At high temperature some of the protons undergo severe changes in linewidths. Those protons that undergo the largest upfield shifts also undergo the most dramatic changes in linewidths. In particular TH1′, TCH3, AH1′, AH2, and TH6 all undergo large changes in linewidths, whereas AH8 and all the H‐2′,2″ protons remain essentially constant. The maximum linewidth occurs at the same temperature for all protons (65°C). This transition does not occur for d(G‐T)·d(C‐A) at 65°C or at any other temperature studied. These changes are cooperative in nature and can be rationalized as a temperature‐induced equilibrium between bound and unbound Cs+, with duplex and single‐stranded DNA. NOE measurements for poly[d(A‐T)] indicate that at high Cs+ the polymer is in a right‐handed B‐conformation. Assignments and NOE effects for the low‐salt 1H spectra of poly[d(A‐T)] agree with those of Assa‐Munt and Kearns [(1984) Biochemistry 23, 791–796] and provide a basis for analysis of the high Cs+ spectra. These results indicate that both polymers adopt a B‐type conformation in both low and high salt. However, a significant variation is the ability of the phosphate backbone to adopt a repeat dependent upon the base sequence. This feature is common to poly[d(G‐T)]·[d(C‐A)], poly[d(A‐T)], and some other pyr–pur polymers [J. S. Cohen, J. B. Wouten & C. L Chatterjee (1981) Biochemistry 20, 3049–3055] but not poly[d(G‐C)].