1388 zyxwvutsrqpon Biochemistry zyxwvut 1980, zyxwvu 19, 1388-1392 Conformational Mobility of Deoxyribonucleic Acid, Transfer Ribonucleic Acid, and Poly( adenylic acid) zyxw As Monitored by Carbon- 1 3 Nuclear Magnetic Resonance Relaxationt Philip H. Bolton* and Thomas L. James* ABSTRACT: The molecular motion of DNA, the native form of tRNA, and partially denatured poly(A) has been investi- gated by carbon- 13 nuclear magnetic resonance zyxwvuts ( 13CNMR). The nuclear Overhauser effect of the RNA samples was measured at 25.1 and 50.3 MHz, and the spin-lattice relax- ation time of all the samples was measured at 50.3 MHz. The NMR data indicate that the local motion of the ribose carbons is much less restricted than that of the bases for DNA and tRNA. The local motion correlation times of the ribose carbons are in the range of 1-7 ns for the samples investigated. T h e role of conformational fluctuations in the biological processes of macromolecules has been gaining recognition during the past few years. The central cellular functions of transcription and translation quite plausibly involve distortion of the conformation of DNA. The packaging of poly- nucleotides into compact forms in chromatin, viruses, and ribosomes involves folding of the nucleic acids. These and other observations indicate that the deformation of poly- nucleotide structure is intimately related to the biological role of nucleic acids. Since the conformational mobility of nucleic acids is related to the energetics of deformation, it is important to have some information concerning the rates and types of molecular motion. Several approaches have been used to monitor the confor- mational fluctuations of nucleic acids. The first information came from hydrodynamic studies which showed that long DNA double helixes are flexible (Eisenberg, 1974). On the average, the angle between base pairs separated by the per- sistence length, - 160 base pairs, is 90’ (Eisenberg, 1974). Examination of the 31PNMR relaxtion of DNA and dou- ble-stranded RNA has given support to the idea that dou- ble-stranded polynucleotides are not rigid rods and that the correlation time for the long-range bending motions of dou- ble-stranded RNA and DNA is on the order of a microsecond (Bolton & James, 1979, 1980). Examination of the rate of decay of the fluorescence an- isotropy of a drug molecule bound to polynucleotides has shown that there is significant local motion of the bases on the time scale of tens of nanoseconds (Wahl et al., 1970; Barkley & Zimm, 1979). 31PNMR studies have indicated that the correlation time for the local motion of the phosphate group of RlVA and DNA is on the order of 0.5 ns (Klevan et al., f From the Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94 143. Received September 27, I979. This research was financially supported by Research Grant GM 25018 from the National Institutes of Health and by Grant RR00892 from the Division of Research Resources, Na- tional Institutes of Health, for maintenance of the UCSF Magnetic Resonance Laboratory. T.L.J. also acknowledges receipt of a Research Career Development Award (AM 00291) from the National Institutes of Health. *Present address: Hall-Atwater Laboratories, Department of Chem- istry, Wesleyan University, Middletown, C T 06457. The local motion correlation times of the different nucleic acids are quite similar with the exception that the 2’ carbon of DNA and poly(A) is apparently less restricted than that for tRNA. The local motion correlation times of the ribose carbons, except perhaps the 2’, do not appear to be strongly coupled to the conformation of the polynucleotide. The 13CNMR results can be combined with those of other investigations to obtain a consistent picture of the internal and overall motions of polynucleotides which have a backbone that is much more flexible than that of the bases. 1979; Bolton & James, 1979, 1980). Thus, there is local motion of polynucleotides which can occur - 1000 times faster than the bending motions which give rise to the persistence length. Of special interest are the rate of conformational fluctuation of the ribose moiety of nucleic acids and the relationship between the mobility of the ribose and that of the bases. It is the conformation of the ribose which is indicative of the overall conformation of a polynucleotide. It is known that DNA can exhibit a wide variety of conformations, but RNA is generally found to exhibit only small deviations from a single conformation. This difference between RNA and DNA may be a manifestation of the conformational mobility of their respective riboses. Examination of the conformation of mo- nonucleosides and mononucleotides, as well as a few small oligomers, leads to the concept of the “rigid nucleotide” (Davies, 1978). However, a theoretical study indicated that the ribose of polynucleotides is conformationally flexible (Levitt & Warshel, 1978), and a preliminary study of the I3C NMR relaxation of DNA showed that the ribose is undergoing fluctuations on the time scale of nanoseconds (Bolton & James, 1979). For the purpose of obtaining more information about the rates and types of conformational fluctuations of the ribose and bases of polynucleotides, the I3C NMR relaxation of DNA, tRNA, and poly(A) has been examined. The poly- nucleotides were chosen to represent a variety of polynucleotide forms to allow investigation of the dependence, if any, of the rates and types of conformational fluctuations of the ribose and bases on the type of polynucleotide. Materials and Methods The poly(A) sample was prepared by dissolving 500 mg of the potassium salt from P-L Biochemicals in 14 mL of 0.1 M NaCI, 2 mM EDTA, and 10 mM H2KP04 at pH 7. The buffer was made up of equal portions of H20 and ’H20. The tRNA sample was prepared by dissolving 500 mg of Sigma type X yeast tRNA in 14 mL of 0.1 M NaCl, 2 mM EDTA, 10 mM MgC12, and 10 mM H2KP04 at pH 7. The buffer was made up of equal portions of H 2 0 and 2H20. Calf thymus DNA from Sigma was briefly sonicated in high salt and dialyzed against 0.1 M NaCl and 10 mM HlKP04 at the pH of the sample. The sample was then diluted with 20% of the 0006-2960/80/0419-1388$01.00/0 0 1980 American Chemical Society