Open states in native polynucleotides

The kinetics for hydrogen exchange at individual base pairs in self-complementary deoxydodecanucleotide duplexes have been estimated from NMR saturation recovery measurements on the resolved imino protons as a function of temperature.The imino protons of dA-dT base pairs in the center of the fully alternating d(C-G-C-G-T-A-T-A-C-G-C-G) duplex exchange a factor of 2-to 3-fold faster than the corresponding protons at the same positions in the partially alternating d(C-G-C-G-A-A-T-T-C-G-C-G) duplex. These exchange parameters are a direct measure of the rate constants for transient opening of individual dA-dT base pairs in the dodecanucleotide duplexes and demonstrate faster opening kinetics for the "TATA" box region compared to the related "AATT" segment.Recent advances in oligonucleotide synthesis have led to the preparation of milligram quantities of DNA fragments of precise sequence containing at least one turn of helix (1). One such example is the self-Scheme I which has been extensively analyzed by single-crystal x-ray analysis in the solid state (2) and high-resolution NMR spectroscopy in aqueous solution (3). Both techniques have established that the central A-T-rich tetranucleotide segment shows conformational polymorphism in the duplex state as a function of temperature and solvent (2, 3).These observations initiated the present experiments, which probe the effect of sequence on the conformation and dynamics of DNA duplexes in solution. The completely alternating self-was synthesized by solid-state methods, and the effect of sequence variations in the center of the duplex was probed by comparative studies of the AATT 12-mer and TATA 12-mer duplexes.Redfield et al. have introduced nuclear Overhauser effect (NOE) and saturation recovery methods to probe the conformation and dynamics of transfer RNA in solution (4). They have demonstrated intra-and inter-base-pair NOEs involving exchangeable and nonexchangeable protons and have correlated the magnitude of the NOE effect with the inverse sixth power dependence of the interproton distance between the saturated and observed resonances. This distance-measuring methodology has wide applications, including its extension to elucidate conformational features of DNA fragments (3), synthetic DNAs (5), and antibiotic-DNA complexes (6) in solution.Hydrogen-deuterium exchange stop-flow measurements have demonstrated that the exchange kinetics of the Watson-Crick imino protons are sensitive markers of the dynamics of base pair opening in the interior of nucleic acid duplexes (7). NMR also can monitor hydrogen exchange, and the early research focused on line width changes of the exchangeable imino protons in the continuous wave mode to evaluate the kinetics of base pair-opening reactions (8, 9). The introduction of Fourier transform methods with H20 solvent suppression (10) readily permitted the incorporation of variable time delays between the saturation and observation pulses to monitor the kinetics of magnetization recovery. The initial measurements were made on...

Section: Bmentioning

confidence: 99%

Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

Patel¹,

Ikuta²,

Kozlowski³

et al. 1983

“…x-r p(x) = o o f(r,s) dr ds [8] [3] in which AS, is the entropy and AfHS the enthalpy of formation of an open segment, R is the gas constant, and T is absolute temperature (13). Because, by hypothesis, each segment contains (L/i) open base pairs and the mean of the excursion is x = f1 -p(x)ldx.…”

mentioning

confidence: 99%

“…The topological nature of such a twist defect leads to stability against rapid decay to the native ground state. The detailed shape of the soliton and the associated diffusive motion will depend upon corresponding details of the potential, V (8). Although the sinusoidal potential of the mechanical model is undoubtedly a great oversimplification of the real potential, it contains the main feature of breaking a bond around 6 = 0.…”

mentioning

confidence: 99%

Nature of the open state in long polynucleotide double helices: possibility of soliton excitations.

Englander¹,

Kallenbach²,

Heeger³

et al. 1980

450

184

The existence of transiently open states in DNA and synthetic polynucleotide double helices as been demonstrated by hydrogen exchange measurements; base pairs reversibly separate and reclose, exposing nucleotide protons to exchange with solvent protons. Recently it has been possible to define the equilibrium kinetic, and activation parameters of the major open state tdat determines base pair hydrogen exchange. However, there is no direct information at the moment about the conformation of the open form. Here we consider the possibility that the low energy and slow opening and closing rates observed reflect a deformation involving several adjacent base pairs. Assuming a mobile open unit capable of diffusing along the double helix, we find that available data are consistent with structures of 10 or so adjacent open pairs. It is further suggested that these structures correspond to thermally induced so on excitations of the double helix, which retain coherence by sharing the energy of a twist deformation among several base tairs. Solitons

“…The rate with which a labile proton exchanges with solvent can be greatly affected by the rate of structural fluctuations that expose such a proton to solvent and therefore can reflect dynamic aspects of conformation (1,2). The most widely used approach to this problem has been the use of 3H/'H exchange gel filtration methods (1,2); model studies with double helical polynucleotides indicate that at low temperatures both imino and amino protons exchange with rates slow enough to be detected by this technique (3)(4)(5)(6)(7). With tRNA, more slowly exchanging protons were observed than could be accounted for by the number of imino and amino hydrogens predicted from the secondary structure, and these extra protons were assigned to tertiary structure (4,(8)(9)(10).…”

mentioning

confidence: 99%

Real-time solvent exchange studies of the imino and amino protons of yeast phenylalanine transfer RNA by Fourier transform NMR.

Johnston¹,

Figueroa²,

Redfield³

1979

Real-time solvent exchange measurements using Fourier transform NMR at 270 MHz are presented. By means of the fast gel filtration column techniques originally developed for tritium exchange experiments, we were able to replace the solvent of a tRNA sample from an 'H20 to an 2H20buffer and obtain a useful spectrum in 2-5 min. At 15'C, there are 5 + 1 lowfield (-11 to -15 ppm relative to 2,2-dimethyl-2-silapentane-5-sulfonate) imino protons with exchange half times of minutes to hours. In addition, the m7G46 C(8) proton and several amino protons are observed to exchange with similar rates. Analogous studies on unfractionated yeast tRNA suggest that such a class of slowly exchanging imino protons is present in several tRNAs, and that the activation energy for exchange is small [t;5 kcal/mol (21 kj/mol)J. We speculate that these imino resonances arise from D-stem protons and that their slow exchange reflects stabilization by the numerous tertiary interactions involving this stem and the Mg2+ bound at the P-10 bend.The measurement of hydrogen exchange rates has become an important tool for studying macromolecular conformation and dynamics. The rate with which a labile proton exchanges with solvent can be greatly affected by the rate of structural fluctuations that expose such a proton to solvent and therefore can reflect dynamic aspects of conformation (1,2). The most widely used approach to this problem has been the use of 3H/'H exchange gel filtration methods (1, 2); model studies with double helical polynucleotides indicate that at low temperatures both imino and amino protons exchange with rates slow enough to be detected by this technique (3-7). With tRNA, more slowly exchanging protons were observed than could be accounted for by the number of imino and amino hydrogens predicted from the secondary structure, and these extra protons were assigned to tertiary structure (4,(8)(9)(10). More recently, stopped-flow ultraviolet spectroscopy has been introduced into hydrogen exchange studies of nucleic acids (11,12). This technique is applicable to much wider rate and temperature ranges than the tritium exchange methods. However, neither of these methods can make an unambiguous assignment of the observed rates to specific hydrogens in the structure.There is a clear NMR spectral differentiation between imino and amino protons of nucleic acids (13), and studies on purified tRNAs have made progress in identifying the imino proton resonances with specific hydrogens in the secondary and tertiary structure (14, 15). Using pulsed Fourier transform NMR (FT NMR), we have studied the relaxation rates of the imino proton resonances of tRNA. At certain temperatures, within about 100C of a melting transition, these rates are a direct measure of out-exchange (16,17), and they are particularly useful in describing the melting transitions in tRNA (18). However, this method cannot be used to measure exchange rates that are less than about 10 sec1 (17).The results presented in this paper extend our measurements of exchange to r...