Internal motions of transfer RNA: a study of exchanging protons by magnetic resonance

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr. After dilution of tRNA-H2O samples in D2O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region. Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CG11 of yeast tRNAPhe at 17 degrees C, or 5 minutes for U8-A14 of yeast tRNA(Asp) at 20 degrees C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five. The exchange rates of other, fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA(Phe) has a lifetime of 14 ms at 17 degrees C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem. Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer. From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether short-lived or long-lived. Buffer effects have also been observed in poly(rA).poly(rU), for which we derive a lifetime of 2.5 ms at 27 degrees C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.     

Proton exchange and base-pair lifetimes in a deoxy-duplex containing a purine-pyrimidine step and in the duplex of inverse sequence

Using proton relaxation and magnetization transfer from water we have measured the imino proton exchange kinetics in two dodecadeoxynucleotide duplexes. One is formed by the self-complementary sequence 5'-d(C-C-T-T-T-C-G-A-A-A-G-G), the other by the inverse sequence. The imino proton exchange rates are found to depend on the concentration of ammonia or imidazole, acting as basic catalysts of proton exchange. Extrapolation of exchange times to infinite catalyst concentration yields the base-pair lifetimes, for instance 40 milliseconds for the central G.C base-pair of the 5'-d(C-C-T-T-T-C-G-A-A-A-G-G) duplex and four milliseconds for its A.T neighbour, at 15 degrees C. These results differ markedly from those reported by other laboratories for similar deoxy compounds. An explanation of the discrepancy has been proposed recently. Differences between base-pair lifetimes indicate that opening is not co-operative. From the catalyst efficiency relative to exchange from isolated nucleosides, we estimate the dissociation constant of each base-pair, e.g. 0.3 x 10(-6) and 1.5 x 10(-5) at 15 degrees C, for the same G.C and A.T base-pairs. The lifetime and dissociation constant of corresponding base-pairs of the two duplexes are similar, except for the central G.C base-pair. This correlates with differences in the solution structures reported by others. We have completed the assignments of the imino protons and of the six cytosine amino protons of the 5'-d(G-G-A-A-A-G-C-T-T-T-C-C) 12-mer. A new base-pair numbering scheme is proposed.     

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.     

Effect of environment, conformation, sequence and base substituents on the imino proton exchange rates in guanine and inosine-containing DNA, RNA, and DNA-RNA duplexes

High-resolution 1H nuclear magnetic resonance in H2O has been used to study the effect of sequence, conformation, environmental factors and base substituents on the exchange behavior of the hydrogen-bonded imino protons of guainine X cytosine and inosine X cytosine base-pairs in DNA, RNA, and DNA-RNA duplexes. The exchange rates were determined by measurement of the spin-lattice relaxation rates of the imino protons as a function of temperature. The exchange was not altered by the presence of high concentrations of salt, and the inability of phosphate to catalyze the exchange indicates that the exchange is limited by formation of a solvent-accessible "open" state. The exchange behavior depends on the duplex conformation and sequence. Exchange from the Z form polymers was orders of magnitude slower than the corresponding duplexes in the B conformation, and the A form RNA duplexes exchanged more slowly than the B form DNA polymers with the same sequence. The exchange behavior of the DNA-RNA hybrids was dependent on whether the purine or the pyrimidine strand contained the deoxyribose sugar. For both the guanine and inosine-containing duplexes, the homopolymer duplexes exchange more slowly than the more stable alternating copolymers. For the alternating duplexes, substitution of cytosine with 5-bromo- or 5-methylcytosine slowed the exchange and increased the activation energy for exchange. The inosine-containing duplexes exchanged more rapidly than the guanosine-containing duplexes, but both showed similar changes in exchange behavior in response to changes in sequence and base substituents. The activation energies for base-pair opening in B form DNA are correlated with the van der Waals contribution to the base-base interaction energy, suggesting that the purine base is partially unstacked in the open state. Using the relaxation measurements to set an upper limit on the exchange rate in poly(dG-dC) and the tritium exchange behavior at low temperature, we find that even though Z-DNA exchanges very slowly, the activation energy is similar to that observed in the A and B form duplexes, suggesting that exchange occurs from a similar open state.     

Premelting thermal fluctuational base pair opening probability of poly(dA).poly(dT) as predicted by the modified self-consistent phonon theory.

We employ a mean field, modified, self-consistent phonon theory to evaluate the single base-pair opening rate and the probability of a base pair in the amino proton exchangeable state for the homopolymer poly(dA).poly(dT) at temperatures below the helix-coil transition region. Our calculated premelting single base-pair opening probabilities are in general agreement with several available experimental estimates from imino proton exchange and formaldehyde-induced DNA melting measurements. These calculated opening probabilities, however, are in disagreement with the prediction of the helix-coil transition theory. Possible reasons for the differences are discussed, especially the possible different definition of a meaningful open state in the premelting region. The premelting open state of the modified self-consistent phonon approximation theory seems to be appropriate to describe a solvent-accessible open configuration that is sufficient to facilitate important chemical reactions such as imino proton exchange and formaldehyde reaction with the bases. This can be compared with the completely unstacked open state of the helix-coil transition theory originally defined in the helix-coil transition region. We propose that the amino proton exchangeable state is different from the open state associated with melting and only involves the breaking of the amino interbase H bond. The agreement between the calculated and experimentally estimated probability of a base pair in the amino proton exchangeable state seems to support this hypothesis.     

Internal Motions of Transfer RNA: A Study of Exchanging Protons by Magnetic Resonance36

Abstract

Proton exchange is a probe of macromolecular structure and kinetics. Its value is enhanced when the exchanging protons can be identified by nmr.

After dilution of tRNA-H₂O samples in D₂O, slowly exchanging imino protons are observed, with exchange times ranging from minutes to days. In many cases they originate from the dihydro-uracil region.

Most slow exchangers are sensitive to buffer catalysis. Extrapolation to infinite buffer concentration yields the life-time of the closed form, in a two-state model of each base-pair. As predicted by the model, the lifetime obtained by extrapolation is independent of the buffer. Typical lifetimes are 14 minutes for CGI 1 of yeast tRNA^Phe at 17°C, or 5 minutes for U8-A14 of yeast tRNA^Asp at 20°C, without magnesium. For most slow exchangers, magnesium increases the lifetime of the closed form, but moderately, by factors never more than five.

The exchange rates of other,fast-exchanging, imino protons, as determined by line-broadening, are found to depend on buffer concentration. Base-pair lifetimes are determined as above. For instance UA6 of yeast tRNA^Phe has a lifetime of 14 ms at 17°C. Base-pairs 4 and 6 have shorter lifetimes than the rest of the acceptor stem.

Imidazole is a good catalyst for proton exchange of both the long-and the short-lived base-pairs, whereas phosphate is not. Tris is efficient except for cases where, possibly, access is impeded by its size; magnesium reduces the efficiency of catalysis by tris buffer.

From the variation of exchange time vs buffer concentration, one determines the buffer concentration for which the exchange rate from the open state is equal to the closing rate. Remarquably, this concentration takes comparable values for most base-pairs, whether shortlived or long-lived.

Buffer effects have also been observed in poly(rA)-poly(rU), for which we derive a lifetime of 2.5 ms at 27°C, and in other polynucleotides. Some of the exchange times identified in the literature as base-pair lifetimes may instead reflect incomplete catalysis.     

Base-pair opening and closing reactions in the double helix. A stopped-flow hydrogen exchange study in poly(rA).poly(rU).

The hydrogen-deuterium exchange of AMP, uridine, poly(rA), and poly(rA) · poly(rU) was investigated by a spectral difference method using stopped-flow spectrophotometry. Proton exchange rates were measured as a function of pH, added catalysts, temperature and salt concentration. The results confirm and extend previous conclusions on the H-exchange chemistry of the bases, on the large equilibrium opening of the double helix, and on its slow opening and closing rates, but an alternative conformation for the major open state is considered. Two H-exchange rate classes are found in poly(rA) · poly(rU). The slower class represents the two exocyclic amino protons of A which exchange through a pre-equilibrium opening mechanism, therefore revealing the fraction of time the helix is open. Base-pairs are open 5% of the time at 25 °C. The faster class is assigned to the U-N-3 H proton, the rate of which is limited by helix opening. Both opening and reclosing of the duplex are slow, 2 s^?1 and 40 s^?1, respectively, at 25 °C. Thermodynamic parameters for the equilibrium helix opening and for the rate of opening were determined. These properties may be consistent with a simple opening involving swinging out of the U base while retaining A more or less stacked within the duplex. The results demonstrate that no faster or more populated helix-open state occurs (when structure is stable). It appears that, unlike opening—closing reactions at a helix end or a helix-coil boundary, internal base opening and closing are innately slow. One implication of this is that any chemical or biological process requiring access to sequences in the interior of a closed stable DNA duplex may be constrained to proceed only on a time scale of seconds, and not in milliseconds or microseconds.     

1H NMR determination of base-pair lifetimes in oligonucleotides containing single base mismatches

Proton nuclear magnetic resonance (NMR) spectroscopy is employed to characterize the kinetics of base-pair opening in a series of 9mer duplexes containing different single base mismatches. The imino protons from the different mismatched, as well as fully matched, duplexes are assigned from the imino-imino region in the WATERGATE NOESY spectra. The exchange kinetics of the imino protons are measured from selective longitudinal relaxation times. In the limit of infinite exchange catalyst concentration, the exchange times of the mismatch imino protons extrapolate to much shorter lifetimes than are commonly observed for an isolated GC base pair. Different mismatches exhibit different orders of base-pair lifetimes, e.g. a TT mismatch has a shorter base-pair lifetime than a GG mismatch. The effect of the mismatch was observed up to a distance of two neighboring base pairs. This indicates that disruption in the duplex caused by the mismatch is quite localized. The overall order of base-pair lifetimes in the selected sequence context of the base pair is GC > GG > AA > CC > AT > TT. Interestingly, the fully matched AT base pair has a shorter base-pair lifetime relative to many of the mismatches. Thus, in any given base pair, the exchange lifetime can exhibit a strong dependence on sequence context. These findings may be relevant to the way mismatch recognition is accomplished by proteins and small molecules.     

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.     

A proton NMR spin-lattice relaxation study of the imino proton exchange kinetics in calf-thymus DNA

The results of semiselective ¹H-nmr inversion recovery experiments on sonicated calf thymus DNA fragments are reported. The measurements were conducted in aqueous solutions containing 85% D₂O, in order to reduce the dipolar contribution to the observed relaxation rates. In solutions containing 0.2M NaCl, 0.4 mM EDTA, and 10 mM cacodylate at pH = 7.0, the exchange rates of the imino protons in A-T base pairs confirm values published earlier in the literature, extrapolating to 0.25 s^?1 at 25°C. Corresponding values for the G-C base pairs are published for the first time, and are about sixfold slower. The addition of up to 0.1M Tris buffer (pH = 7.3 at 25°C), caused a striking increase in the measured exchange rates for both the A-T and G-C imino protons, resembling the effect recently observed for poly(rA)-poly(rU) and poly(rI)-poly(rC), and suggesting that the exchange rates measured for nucleic acid duplexes in low buffer concentrations at neutral pH do not reflect base-pair opening rates as assumed in the past. Lower limits to the base-pair opening rates could be estimated from extrapolation of the experimental data to infinite buffer concentration, and are 1 × 10³ s^?1 for the A-T, and 50 s^?1 for the G-C, base paris at 62°C.     

Imino proton exchange and base-pair kinetics in RNA duplexes

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino proton, we have measured the base-pair lifetimes and the dissociation constants of six RNA duplexes: [r(CGCGAUCGCG)](2), [r(CGCGAAUUCGCG)](2), [r(CCUUUCGAAAGG)](2), [r(CGCACGUGCG)](2), [r(GGU(8)CC).r(GGA(8)CC)], and [poly(rA).poly(rU)], and we compare them with those of their DNA homologues. As predicted by a two-state (closed/open) model of the pair, the imino proton exchange times decrease linearly vs. the inverse of catalyst concentration. As in DNA duplexes, base pairs open one at a time, and the kinetics is in most cases insensitive to the nature of the adjacent residues. The lifetime of the r(G.C) pairs, 40 to 50 ms, is longer than that of the equivalent in the corresponding oligodeoxynucleotides, and the dissociation constants, about 10(-)(7), are slightly smaller. The r(A.U) opening and closing rates are much larger than those of the d(A.T) pairs, but the stabilities are comparable.     

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).     

Proton exchange in DNA-luzopeptin and DNA-echinomycin bisintercalation complexes: rates and processes of base-pair opening.

Imino proton exchange studies are reported on the complexes formed by bisintercalation of luzopeptin around the two central A.T pairs of the d(CCCATGGG) and d(AGCATGCT) duplexes and of echinomycin around the two central C.G pairs of the d(AAACGTTT) and d(CCAAACGTTTGG) duplexes. The depsipeptide backbone of the drugs occupies the minor groove of the complexes at the bisintercalation site. The exchange time of the amide protons of the depsipeptide rings provides a lower estimate of the complex lifetime: 20 min at 15 degrees C for the echinomycin complexes and 4 days at 45 degrees C for the luzopeptin complexes. The exchange time of imino protons is always shorter than the complex lifetime. Hence, base pairs open even within the complexed oligomers. For the two base pairs sandwiched between the aromatic rings of the drug, the base-pair lifetime is strongly increased, and the dissociation constant is correspondingly reduced. Hence, the lifetime of the open state is unchanged. This suggests similar open states in the free duplex and in the complex. In contrast to the sandwiched base pairs, the base pairs flanking the intercalation site are not stabilized in the complex. Thus, the action of the bisintercalating drug may be compared to a vise clamping the inner base pairs. Analysis suggests that base-pair opening may require prior unwinding or bending of the DNA duplex.     

Processes of base-pair opening and proton exchange in Z-DNA

Using proton magnetic resonance, we have investigated imino and amino proton exchange in the Z form of the four oligomers d(Cbr8GCGCbr8G), d(CGm5CGCG), d(CG)6, and d(CG)12. In the latter two oligomers, all five exchangeable protons have been assigned. We find that proton acceptors such as NH3 or the basic form of Tris enhance imino proton exchange. The base-pair lifetime can then be obtained by extrapolation of the exchange time to infinite concentration of proton acceptor. For d(CG)6 and d(CG)12, the values are ca. 3.5 ms at 80 degrees C and ca. 130 ms at 35 degrees C. The latter value is about 65 times longer than in the same oligomers in the B form. The activation energy of base-pair opening, 80 kJ/mol, is the same in the Z and the B forms of d(CG)12. At 5 degrees C, the base-pair lifetime is about 3 s, much smaller than the time constant of the Z to B transition, to which it is therefore unrelated. The base-pair dissociation constant at 35 degrees C, 0.5 X 10(-6), is 5 times smaller than for the same oligomers in the B form. In the absence of added catalyst, at pH 7, the exchange time of the imino proton is 30 min at 5 degrees C. That of both cytidine amino protons, assigned by NOE, is about 50 min. The longest proton exchange time, ca. 330 min, is assigned unambiguously to the guanosine amino protons. Thus assigned and interpreted in terms of exchange chemistry rather than structural kinetics, the exchange times do not support earlier models of Z-DNA internal motions.     

NMR studies on oligodeoxyribonucleotides containing the dam methylation site GATC. Comparison between d(GGATCC) and d(GGm6ATCC)

The conformation of two hexanucleotides, d(GGATCC) and d(GGm6ATCC), has been studied by proton nuclear magnetic resonance. Nuclear Overhauser effect (NOE) measurements on d(GGATCC) are in agreement with a normal B form right-handed helical structure. The single- and double-strand resonances are in fast exchange on a proton NMR time scale. The exchange is observed to be slow for d(GGm6ATCC); up to the Tm, separate resonances are observed for each state, though above the Tm exchange becomes more rapid. The preferred orientation of the adenosine methylamino group (methyl cis to N1) hinders base-pair formation. At 0 degree C irradiation of the m6A-T imino proton gives an NOE to AH2, showing that base pairing is Watson-Crick. Intra- and interresidue NOEs show that the helix is right handed and in the B form. Comparing results on the two oligomers demonstrates that adenosine methylation induces little or no change in the conformation of the helix but reduces the Tm from 45 to 32 degrees C. All of the amino proton resonances, as well as the imino resonances, have been assigned. From NOE experiments on the unmethylated oligomer we have located the Watson-Crick and non-Watson-Crick adenosine amino protons. At 0 degree C these resonances show broadening due to rotation of the amino group, and their rotation is slightly slower than for the adjacent guanosine amino group, though both these amino groups have lifetimes of less than 10 ms at 0 degree C. The imino protons show normal behavior, disappearing from the spectra ca. 20 degrees C below the Tm.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H nuclear magnetic resonance study of the dynamic properties of the B and Z-forms of poly[d(A-br5C).d(G-T)]

Poly[d(A-br5C).d(G-T)], a synthetic polynucleotide with a 50% A-T base composition, undergoes a reversible, highly co-operative transition between the right-handed B and left-handed Z conformations. The latter is stabilized at both elevated temperature and ionic strength. The B and Z-forms of poly[d(A-br5C).d(G-T)] coexist in 4.6 M-NaCl at 45 degrees C. Due to slow exchange, two sets of Tim and Gim resonances are observed and can be assigned to the B and Z conformations (the chemical shifts are, respectively, Tim = 13.4, 14.1 p.p.m. (parts/million); and Gim = 11.9, 12.4 p.p.m.). Measurements of the 1H spin-lattice (R1) and spin-spin (R2) relaxation rates of the exchangeable thymine (Tim) and guanine (Gim) imino protons have been used to probe the internal dynamics of the B and Z-forms of poly[d(A-br5C).d(G-T)] and the mechanism of the B-Z transition. The proton exchange behavior in the B and Z conformations is quite different. At elevated temperature, R1 for both Tim and Gim in the B conformation is dominated by exchange with the solvent, with Tim exchanging more rapidly than Gim. This demonstrates that exchange involves the opening of single base-pairs and that neighboring A-T and G-br5C base-pairs exchange independently of each other. B-form poly[d(A-br5C).d(G-T)] is unusual in that there is an acceleration of the Tim exchange rate with increasing NaCl concentration. Conversion to the Z-form by addition of 4.5 M-NaCl dramatically reduces both the Tim and Gim exchange rates (estimated to be less than 2 s-1 at 70 degrees C). Thus, the G-br5C base-pair and, in particular, the A-T base-pair are stabilized in the Z conformation. By measuring relaxation rates at 45 to 50 degrees C where the B and Z-forms are in equilibrium, we find that the B-Z interconversion rates are less than two per second. In the B conformation at 25 degrees C, the dipolar contributions to the imino proton relaxation rates are about one-third of those expected on the basis of a rigid rod model for 65 base-pair fragments, a difference we assign to large amplitude (30 degrees high frequency (less than 100 ns) out-of-plane motions of the bases. Conversion to the Z conformation has little effect on the dipolar contributions to relaxation, i.e. on the internal motions.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG)

The nonsymmetrical double-helical hybrid dodecamer d(CGTTATAATGCG).r(CGCAUUAUAACG) was synthesized with solid-phase phosphoramidite methods and studied by high-resolution 2D NMR. The imino protons were assigned by one-dimensional nuclear Overhauser methods. All the base protons and H1', H2', H2", H3', and H4' sugar protons of the DNA strand and the base protons, H1', H2', and most of the H3'-H4' protons of the RNA strand were assigned by 2D NMR techniques. The well-resolved spectra allowed a qualitative analysis of relative proton-proton distances in both strands of the dodecamer. The chemical shifts of the hybrid duplex were compared to those of the pure DNA double helix with the same sequence (Wemmer et al., 1984). The intrastrand and cross-strand NOEs from adenine H2 to H1' resonances of neighboring base pairs exhibited characteristic patterns that were very useful for checking the spectral assignments, and their highly nonsymmetric nature reveals that the conformations of the two strands are quite different. Detailed analysis of the NOESY and COSY spectra, as well as the chemical shift data, indicate that the RNA strand assumes a normal A-type conformation (C3'-endo) whereas the DNA strand is in the general S domain but not exactly in the normal C2'-endo conformation. The overall structure of this RNA-DNA duplex is different from that reported for hybrid duplexes in solution by other groups (Reid et al., 1983a; Gupta et al., 1985) and is closer to the C3'-endo-C2'-endo hybrid found in poly(dA).poly(dT) and poly(rU).poly(dA) in the fiber state (Arnott et al., 1983, 1986).     

Proton exchange and base-pair opening kinetics in 5''-d(CGCGAATTCGCG)-3'' and related dodecamers.

We have used nuclear magnetic resonance (NMR) spectroscopy to measure the lifetimes of individual base-pairs in the palindromic DNA oligonucleotide 5'-d(CGCGAATTCGCG)-3' and in three other dodecamers with symmetrical base substitutions in the sites underlined. The resonances of the hydrogen-bonded imino protons in each of the substituted oligomers in the duplex form have been assigned using one dimensional nuclear Overhauser effect (1-D NOE) experiments. The lifetimes have been obtained from the dependence of selective longitudinal relaxation times and linewidths of the imino proton resonances on the concentration of base catalyst (Tris) at 25 degrees C and in the presence of 50 mM NaCl. The lifetimes of the central A.T base-pairs have been found to depend on base sequence. They are greatly increased in the dodecamer 5'-d(CGCAAATTTGCG)-3' which contains an A3T3 tract. The lifetimes of the central A.T base-pairs in 5'-d(CGCGAATTCGCG)-3', 5'-d(CGCTAATTAGCG)-3' and 5'-d(CGCCAATTGGCG)-3' are comparable. In all dodecamers, the lifetime of the A.T base-pair at the 5'-end of the AnTn tract is the shortest. The anomalous opening kinetics of the A.T base-pairs can be correlated to the bending properties of the corresponding sequences.     

Study of structure, base-pair opening kinetics and proton exchange mechanism of the d-(AATTGCAATT) self-complementary oligodeoxynucleotide in solution.

Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.