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High resolution nuclear magnetic resonance (NMR) and ethidium bromide binding studies are used to demonstrate that poly d(G-T) forms an ordered double helical structure at low temperatures (below 24 degrees C in 0.3 M NaCl) in which G and T are hydrogen bonded together in a wobble base pair hydrogen bonding scheme as proposed earlier by Lezius and Domin. Alternative hydrogen bonding schemes involving the tautomeric form of either T or G, such as have been proposed to account for mutation rates in DNA synthesis, are eliminated.

In this paper we report successful observations of proton NMR spectra of native double helical salmon sperm and calf thymus DNA of various lengths. Measurements of the linewidths arising from proton-proton dipolar interactions are used to obtain information about the dynamic behavior of DNA helices in solution. Depending upon which protons are used to monitor the local internal motions of the DNA, different results are obtained. The lowfield resonances from hydrogen-bonded imino protons in the base pairs indicate that the correlation time for reorientation of base pairs is less than 3 X 10-7 sec, whereas correlation times for motion of neighboring sugar protons relative to the aromatic protons are <5 X 10-8 sec for DNA that is over 200 base pairs long. These observations indicate that there is considerably more internal flexibility in the DNA molecules, especially in the backbone, than is indicated by classic hydrodynamic studies of DNA.The determination of the conformational state and the flexibility of DNA is of considerable interest in connection with studies of the hydrodynamic properties of DNA (1-4), the structure of DNA in chromosomes (5-8), the folding of DNA in phage particles (9), supercoiling in circular DNA (10, 11) and linear DNA (12), and computations of superhelical structure and energetics of DNA foldings (5-8, 10, 11). Information on conformational fluctuations in DNA is also of interest in connection with the use of spectroscopic tools (circular dichroism, infrared, Raman) to deduce the average conformational state of DNA (13)(14)(15).NMR spectroscopy is an excellent tool for studying flexibility in large molecules. Some preliminary 31P NMR studies of high molecular weight calf thymus DNA and chromatin (16) and nucleosome core particles have been reported (17,18), and these indicate there is considerable flexibility in the backbone (linewidths of 20-50 Hz). Previous attempts to use 1H (19) and '3C (20) NMR on native DNA were unsuccessful. Patel has successfully studied the 'H NMR of simple sequence synthetic DNA (21,22), but these molecules are believed to be unusually flexible due to formation of branched duplex regions and hairpins and therefore may not be good models for studying flexibility in native DNA. In 1977, we demonstrated (23) that it is possible to obtain partially resolved NMR spectra on high molecular weight native double-helical DNA, and in the present paper we have used NMR and the fact that NMR linewidths and relaxation times are sensitive to local internal motions to detect flexibility in the DNA that is not evident when the DNA is studied by other classical techniques.Our results show that it is possible to obtain useful proton NMR spectra on DNA of unexpectedly high molecular weight and make possible a number of applications of NMR to the study of DNA properties in solution. EXPERIMENTAL Salmon sperm (or calf thymus) DNA (Worthington) was dissolved (10 mg/ml) in 100 ml of 0.1 M NaAcO/2 mM ZnCl2 at pH 4.6, and S1 nuclease (1000 international units) was added. This so...

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