Deoxyguanosine-deoxyadenosine pairing in the d(C-G-A-G-A-A-T-T-C-G-C-G) duplex: conformation and dynamics at and adjacent to the dG X dA mismatch site

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d-(C1-G2-A3-G4-A5-A6-T6-T5-C4-G3-C2-G1) self-complementary dodecanucleotide (henceforth called 12-mer GA) that contains a dG X dA purine-purine mismatch at position 3 in the sequence. These results are compared with the corresponding d(C-G-C-G-A-A-T-T-C-G-C-G) dodecamer duplex (henceforth called 12-mer) containing standard Watson-Crick base pairs at position 3 [Patel, D.J., Kozlowski, S.A., Marky, L.A., Broka, C., Rice, J.A., Itakura, K., & Breslauer, K.J. (1982) Biochemistry 21, 428-436]. The dG X dA interaction at position 3 was monitored at the guanosine exchangeable H-1 and nonexchangeable H-8 protons and the nonexchangeable adenosine H-2 proton. We demonstrate base-pair formation between anti orientations of the guanosine and adenosine rings on the basis of nuclear Overhauser effects (NOE) observed between the H-2 proton of adenosine 3 and the imino protons of guanosine 3 (intra base pair) and guanosines 2 and 4 (inter base pair). The dG(anti) X dA(anti) pairing should result in hydrogen-bond formation between the guanosine imino H-1 and carbonyl O-6 groups and the adenosine N-1 and NH2-6 groups, respectively. The base pairing on either side of the dG X dA pair remains intact at low temperature, but these dG X dC pairs at positions 2 and 4 are kinetically destabilized in the 12-mer GA compared to the 12-mer duplex. We have estimated the hydrogen exchange kinetics at positions 4-6 from saturation-recovery measurements on the imino protons of the 12-mer GA duplex between 5 and 40 degrees C. The measured activation energies for imino proton exchange in the 12-mer GA are larger by a factor of approximately 2 compared to the corresponding values in the 12-mer duplex. This implies that hydrogen exchange in the 12-mer GA duplex results from a cooperative transition involving exchange of several base pairs as was previously reported for the 12-mer containing a G X T wobble pair at position 3 [Pardi, A., Morden, K.M., Patel, D.J., & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574]. We have assigned the nonexchangeable base protons by intra and inter base pair NOE experiments and monitored these assigned markers through the 12-mer GA duplex to strand transition.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of the thymine C5 methyl group on spontaneous base pair breathing in DNA

Sequences of four or more AT base pairs without a 5'-TA-3' step, so-called A-tracts, influence the global properties of DNA by causing curvature of the helix axis if phased with the helical repeat and also influence nucleosome packaging. Hence it is interesting to understand this phenomenon on the molecular level, and numerous studies have been devoted to investigations of dynamical and structural features of A-tract DNA. It was early observed that anomalously slow base pair-opening kinetics were a striking physical property unique to DNA A-tracts (Leroy, J. L., Charretier, E., Kochoyan, M., and Gueron, M. (1988) Biochemistry 27, 8894-8898). Furthermore, a strong correlation between DNA curvature and anomalously slow base pair-opening dynamics was found. In the present work it is shown, using imino proton exchange measurements by NMR spectroscopy that the main contribution to the dampening of the base pair-opening fluctuations in A-tracts comes from the C5 methylation of the thymine base. Because the methyl group has been shown to have a very limited effect on the DNA curvature as well as the structure of the DNA helix, the thymine C5 methyl group stabilizes the helix directly. Empirical potential energy calculations show that methylation of the tract improves the stacking energy of a base pair with its neighbors in the tract by 3-4 kcal/mol.     

Physical studies of 5S RNA variants at position 66.

Two variants of the 5S RNA of E. coli have been examined by imino proton NMR spectroscopy, one of them a deletion of A66 (Christiansen, J., Douthwaite, S.R., Christensen, A. and Garrett, R.A. (1985) EMBO J. 4, 1019-1024) and the other a replacement of A66 with a C (Goringer, H.U. and Wagner, R. (1986) Biol. Chem. Hoppe-Seyler 367, 769-780). Both are of interest because the role the bulged A in helix II of 5S RNA is supposed to play in interactions with ribosomal protein L18. The data show that the structural perturbations that result from these mutations are minimal, and assign the resonances of some of the imino protons around position 66. Some mutations at or near position 66 greatly reduce the L18-dependent increase in the circular dichroism of 5S RNA at 267 nm first observed by Bear and coworkers (Bear, D.G., Schleich, T., Noller, H.F. and Garrett, R.A. (1977) Nucl. Acids Res. 4, 2511-2526).     

Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G)

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poly(dA-dT).poly(dA-dT) two-pathway proton exchange mechanism. Effect of general and specific base catalysis on deuteration rates

The deuteration rates of the poly(dA-dT).poly(dA-dT) amino and imino protons have been measured with stopped-flow spectrophotometry as a function of general and specific base catalyst concentration. Two proton exchange classes are found with time constants differing by a factor of 10 (4 and 0.4 s-1). The slower class represents the exchange of the adenine amino protons whereas the proton of the faster class has been assigned to the thymine imino proton. The exchange rates of these two classes of protons are independent of general and specific base catalyst concentration. This very characteristic behavior demonstrates that in our experimental conditions the exchange rates of the imino and amino protons in poly(dA-dT).poly(dA-dT) are limited by two different conformational fluctuations. We present a three-state exchange mechanism accounting for our experimental results.     

NMR analysis of helix I from the 5S RNA of Escherichia coli.

The structure of helix I of the 5S rRNA from Escherichia coli has been determined using a nucleolytic digest fragment of the intact molecule. The fragment analyzed, which corresponds to bases (-1)-11 and 108-120 of intact 5S rRNA, contains a G-U pair and has unpaired bases at its termini. Its proton resonances were assigned by two-dimensional NMR methods, and both NOE distance and coupling constant information have been used to calculate structural models for it using the full relaxation matrix algorithm of the molecular dynamics program XPLOR. Helix I has A-type helical geometry, as expected. Its most striking departure from regular helical geometry occurs at its G-U, which stacks on the base pair to the 5' side of its G but not on the base pair to its 3' side. This stacking pattern maximizes interstrand guanine-guanine interactions and explains why the G-U in question fails to give imino proton NOE's to the base pair to 5' side of its G. These results are consistent with the crystal structures that have been obtained for wobble base pairs in tRNAPhe [Mizuno, H., & Sundaralingam, M. (1978) Nucleic Acids Res. 5, 4451-4461] and A-form DNA [Rabbinovich, D., Haran, T., Eisenstein, M., & Shakked, Z. (1988) J. Mol. Biol. 200, 151-161]. The conformations of the terminal residues of helix I, which corresponds to bases (-1)-11 and 108-120 of native 5S RNA, are less well-determined, and their sugar puckers are intermediate between C2' and C3'-endo, on average.     

Assignment of resonances in the Escherichia coli 5 S RNA fragment proton NMR spectrum using uniform nitrogen-15 enrichment

The downfield proton NMR spectrum of aqueous uniformly nitrogen-15 enriched 5 S RNA fragment is presented. Selective nitrogen-15 decoupling difference proton spectroscopy revealed nitrogen-15 chemical shifts of fragment imino nitrogens. Nitrogen chemical shifts of nucleic acid guanine and uracil imino nitrogens have separate small ranges. Nitrogen-15 and proton chemical shift correlation by the heteronuclear decoupling permitted the identification of the base type of some previously unassigned imino proton resonances in the 5 S RNA fragment spectrum. Corresponding resonances in the natural isotopic abundance 5 S RNA fragment spectrum are assigned to base types by comparison with the enriched sample spectrum.     

Influence of the polyamines spermine and spermidine on yeast tRNAPhe as revealed from its imino proton NMR spectrum.

A comparison of imino proton NMR spectra of yeast tRNAPhe recorded at various solution conditions indicates, that polyamines have a limited effect on the structure of this tRNA molecule. Polyamines are found to catalyse the solvent exchange of several imino protons in yeast tRNAPhe not only of non hydrogen bonded imino protons, but also of imino protons of the GU and of some AU and tertiary base pairs. It is concluded that at low levels of catalysing components the exchange rates of the latter protons are not determined by the base pair lifetime. In the presence of high levels of spermidine the solvent exchange rates of imino protons of several base pairs in the molecule were assessed as a function of the temperature. Apparent activation energies derived from these rates were found to be less than 80 kJ/mol, which is indicative for (transient) independent opening of the corresponding base pairs. In the acceptor helix the GU base pair acts as a dynamic dislocation. The AU base pairs at one side of the GU base pair exhibit faster transient opening than the GC base pairs on the other side of this wobble pair. The base pairs m2GC10 and GC11 from the D stem and GC28 from the anticodon stem show relatively slow opening up to high temperatures. Model studies suggest that 1-methyladenosine, an element of tRNA itself, catalyses imino proton solvent exchange in a way similar to polyamines.     

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation.

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance investigation of the conformation and dynamics in the synthetic deoxyribonucleic acid decamers d(ATATCGATAT) and d(ATATGCATAT)

A variety of one-dimensional proton NMR methods have been used to investigate the properties of two synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of two-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential "melting" of the A . T base pairs and by comparison of the spectra of the two decamers. Below 20 degree C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A X T and G X C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52 degrees C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8, and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5'ApT3'). The base protons (AH8, GH8, and TH6) appear to be located close (1.9-2.3 A) to sugar H2',2" protons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU)

The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.     

Variation of nonexchangeable proton resonance chemical shifts as a probe of aberrant base pair formation in DNA

Variation of nonexchangeable proton resonance chemical shifts for deoxycytidine and deoxy-adenosine as a function of protonation and imino tautomer formation has been determined. Protonation induces downfield shifts of proton resonances whereas formation of the rare imino tautomer induces upfield shifts. Titration curves are constructed on the basis of spectrophotometrically determined pK values. Excellent fit is obtained between theoretical titration curves and experimental data, which indicates that chemical shifts of base protons may be used to quantitatively determine the relative concentrations of either rare imino tautomeric conformations or protonated base forms. These data may be utilized as an aid in the elucidation of the nature of hydrogen bonding between mismatched base pairs in DNA oligomers containing cytosine or adenine residues. These data, in conjunction with the oligonucleotide study of Patel et al. [Patel, D. J., Kozlowski, S.A., Ikuta, S., & Itakura, K. (1984) Biochemistry 23, 3218-3226], have been used to rigorously argue the existence of a "protonated" adenine residue in the A-C mismatch. This structure allows reconciliation of the NMR solution data with crystallographic data [Hunter, W.N., Brown, T., Anand, N.N., & Kennard, O. (1986) Nature (London) 320, 552-555], which support the protonated base pair.     

High base pair opening rates in tracts of GC base pairs

Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.     

NMR spectroscopy of RNA duplexes containing pseudouridine in supercooled water

We have performed NMR experiments in supercooled water in order to decrease the temperature-dependent exchange of protons in RNA duplexes. NMR spectra of aqueous samples of RNA in bundles of narrow capillaries that were acquired at temperatures as low as -18 degrees C reveal resonances of exchangeable protons not seen at higher temperatures. In particular, we detected the imino protons of terminal base pairs and the imino proton of a non-base-paired pseudouridine in a duplex representing the eukaryotic pre-mRNA branch site helix. Analysis of the temperature dependence of chemical shift changes (thermal coefficients) for imino protons corroborated hydrogen bonding patterns observed in the NMR-derived structural model of the branch site helix. The ability to observe non-base-paired imino protons of RNA is of significant value in structure determination of RNA motifs containing loop and bulge regions.     

Properties of pseudouridine N1 imino protons located in the major groove of an A-form RNA duplex.

The exchangeable N1 imino protons of two pseudouridine (psi) bases located at adjacent internal positions within an undecamer RNA duplex (5'AUAC psi psi ACCUG/3'UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The psi N1 imino protons of these residues (which are not involved in interstrand Watson-Crick hydrogen bonding) are protected from chemical exchange with the solvent water and thus are observable in the proton NMR spectrum in H2O (1). These protons will exchange readily at increased pH values or upon thermal denaturation of the duplex. The longitudinal (T1) relaxation times of the psi N1 imino protons in 100 mM NaCl or in 10 mM MgCl2 and 100 mM NaCl are approximately two-fold faster than those of the psi N3 imino protons which are involved in Watson-Crick base pairing. With the addition of spermidine, the psi N1 imino protons become readily exchangeable at a temperature some 20 degrees C below the melting temperature of the duplex.     

Polarity of annealing and structural analysis of the RNase H resistant alpha-5'-d[TACACA].beta-5'-r[AUGUGU] hybrid determined by high-field 1H, 13C, and 31P NMR analysis.

The novel hybrid duplex alpha-5'-d[TACACA]-3'.beta-5'-r[AUGUGU]-3' was analyzed extensively by 1D and 2D NMR methods. Two forms of the duplex exist in about an 80:20 ratio. Analysis of the exchangeable imino protons of the major component revealed that three AU and one AT base pair are present in addition to two GC base pairs, confirming that the duplex anneals in parallel orientation. The presence of the AT base pair, which can only be accounted for by a parallel duplex, was confirmed by a selective INEPT experiment, which correlated the thymidine imino proton to its C5 carbon. The lesser antiparallel form could be detected by exchangeable and nonexchangeable proton resonances in both strands. An exchange peak was observed in the NOESY spectrum for the thymidine methyl group resonance in both the predominant and lesser conformations, indicating the lifetime of the individual structures was on the millisecond time scale. The nonexchangeable protons of the predominant duplex were assigned by standard methods. The sugar pucker of the ribonucleosides was determined to be of the "S" type by a pseudorotation analysis according to Altona, with the J-couplings measured from the multiplet components of the phase-sensitive COSY experiment. The NOE pattern observed for the alpha-deoxynucleosides also suggested an S-type sugar pucker. The adoption of an S-type sugar pucker for both strands indicates that, in contrast to RNA.DNA duplexes formed exclusively from beta-nucleotides, the alpha-DNA.beta-RNA duplex may form a B-type helix. The 31P resonances of the alpha and beta strands have very different chemical shifts in the hybrid duplex and the difference persists above the helix melting temperature, indicating an intrinsic difference in 31P chemical shift for nucleotides differing only in the configuration about the glycosidic bond.     

Alteration of A.T base-pair opening kinetics by the ammonium cation in DNA A-tracts

We have investigated by NMR the effects of NH(4)(+) on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)](2) and [d(GCA(4)T(4)GC)](2), and of a B-DNA duplex,[d(CGCGATCGCG)](2). Upon NH(4)(+) addition to [d(CGCGAATTCGCG)](2), the adenosine H2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G.C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA(4)T(4)GC)](2) and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)](2). The localization of the shifted protons suggests an effect related to NH(4)(+) binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH(4)(+) concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH(4)(+) binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH(4)(+) concentrations show the occurrence of two native conformations in proportions depending on the NH(4)(+) concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH(4)(+) concentration is kept larger than 1 M, the A.T imino proton exchange times of A-tract sequences exhibit a linear dependence versus the inverse of the NH(3) proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (W?rml?nder, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615).     

Assignment of resonances in the downfield proton spectrum of Escherichia coli 5S RNA and its nucleoprotein complexes using components of a ribonuclease-resistant fragment

The downfield (9-15 ppm) proton NMR spectra of oligonucleotides derived from the ribonuclease A resistant fragment of Escherichia coli 5S RNA have been examined in aqueous solution at 500 MHz. Comparison of these spectra with those of the 5S RNA fragment and intact 5S RNA using both chemical shift and nuclear Overhauser enhancement effect criteria indicates that several aspects of 5S RNA secondary structure are also present in the structures assumed in solution by these much smaller molecules. Analysis of these spectra permits the assignment of some imino proton resonances which could not be assigned with certainty on the basis of NMR data previously obtained on intact 5S RNA or its nucleoprotein complexes. Several previous resonance assignments are confirmed. Studies on oligonucleotide components of fragment and a reconstituted fragment show that at least two conformations of the procaryotic loop exist.