Resonance assignment of the 500-MHz proton NMR spectrum of self-complementary dodecanucleotide d-GGATCCGGATCC: altered conformations at BamHI cleavage sites

Resonance assignments of nonexchangeable base and sugar protons of the self-complementary dodecanucleotide d-GGATCCGGATCC have been obtained by two-dimensional NMR methods and strategies derived from interproton distance calculations on different secondary structures of nucleic acids. Conformational details about the glycosidic dihedral angle and sugar pucker have been derived from the relative intensities of cross peaks in the two-dimensional J-correlated and nuclear Overhauser enhancement correlated spectra in D2O solution. It is observed that d-GGATCCGGATCC assumes a predominantly B-type conformation with sequence-dependent changes along the chain. The recognition site of BamHI shows a distinctly different geometrical environment. The sugar rings of G1 and G7 assume a C3'-endo geometry while the rest of the sugars possess C2'-endo geometry.     

Sequence-specific recognition of DNA: assignment of nonexchangeable proton resonances in the consensus Pribnow promoter DNA sequence by two-dimensional NMR

The resonances of most of the nonexchangeable protons of both + and - strands of the consensus Pribnow dodecamer d( CGTTATAATGCG ) have been assigned by two-dimensional nuclear magnetic resonance methods. Application of the two-dimensional nuclear Overhauser effect ( NOESY ) sequential connectivity method, combined with two-dimensional autocorrelated ( COSY ) spectra to reveal scalar-coupled protons, results in assignment of virtually all of the base and sugar protons, except the sugar C5 protons which are inadequately resolved. Analysis of the nuclear Overhauser data indicates that the helix assumes a fairly uniform B form conformation.     

Sequential resonance assignment of the 500-MHz nmr spectrum of d-(CG)6 and its structure under low-salt conditions

Two-dimensional (2D) nmr methods (correlated spectroscopy, nuclear Overhauser enhancement spectroscopy, and relayed correlated spectroscopy) have been used to obtain resonance assignment of the nonexchangeable base and sugar protons of a double-helical DNA segment, d-(CG)₆ in D₂O solutions under conditions of low ionic strength. Detailed information about the glycosidic torsion angle, sugar geometry, stacking patterns of the bases, and the overall solution structure of the dodecanucleotide has been obtained from the relative intensities of cross-peaks in the 2D spectra. The molecule shows general features of B-DNA under the experimental conditions employed. However, in spite of the repeating base sequence, there are subtle and detectable variations in the structure along the double helix. The terminal residues show considerable conformational flexibility.     

Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.     

Sequence-dependent conformations of DNA duplexes: the TATA segment of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

The base and sugar protons of the d(G-G-T-A-T-A-C-C) duplex have been assigned from two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements in D₂O solution at 25°C. The nucleic acid protons have been assigned from NOEs between protons on adjacent bases on the same and partner strands, as well as from NOEs between the base protons and their own and 5′-flanking H1′, H2′, H2″, H3′, and H4′ sugar protons. These assignments are confirmed from coupling constant and NOE connectivities within the sugar protons of a given residue. Several of these NOEs exhibit directionality and demonstrate that the d(G-G-T-A-T-A-C-C) duplex is a right-handed helix. The relative magnitude of the NOEs between the base protons and the sugar H2′ protons of its own and 5′-flanking sugar demonstrate that the TATA segment of the d(G-G-T-A-T-A-C-C) duplex adopts a B-DNA type helix geometry in solution, in contrast to the previous observation of a A-type helix for the same octanucleotide duplex in the crystalline state.     

Novel solution conformation of DNA observed in d(GAATTCGAATTC) by two-dimensional NMR spectroscopy

Resonance assignments of nonexchangeable base and sugar protons of the self-complementary dodecanucleotide d(GAATTCGAATTC) have been obtained by using the two-dimensional Fourier transform NMR methods correlated spectroscopy and nuclear Overhauser effect spectroscopy. Conformational details about the sugar pucker, the glycosidic dihedral angle, and the overall secondary structure of the molecule have been derived from the relative intensities of cross peaks in the two-dimensional NMR spectra in aqueous solution. It is observed that d(GAATTCGAATTC) assumes a novel double-helical structure. The solution conformations of the two complementary strands are identical, unlike those observed in a related sequence in the solid state. Most of the five-membered sugar rings adopt an unusual O1'-endo geometry. All the glycosidic dihedral angles are in the anti domain. The AATT segments A2-T5 and A8-T11 show better stacking compared to the rest of the molecule. These features fit into a right-handed DNA model for the above two segments, with the sugar geometries different from the conventional ones. There are important structural variations in the central TCG portion, which is known to show preferences for DNase I activity, and between G1-A2 and G7-A8, which are cleavage points in the EcoRI recognition sequence. The sugar puckers for G1 and G7 are significantly different from the rest of the molecule. Further, in the three segments mentioned above, the sugar phosphate geometry is such that the distances between protons on adjacent nucleotides are much larger than those expected for a right-handed DNA. We suggest that such crevices in the DNA structure may act as "hot points" in initiation of protein recognition.     

Conformation and dynamics of the Pribnow box region of the self-complementary d(C-G-A-T-T-A-T-A-A-T-C-G) duplex in solution

Nuclear magnetic resonance (NMR) has been used to monitor the conformation and dynamics of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called Pribnow 12-mer), which contains a TATAAT Pribnow box and a central core of eight dA X dT base pairs. The exchangeable imino and nonexchangeable base protons have been assigned from one-dimensional intra and inter base pair nuclear Overhauser effect (NOE) measurements. Premelting conformational changes are observed at all the dA X dT base pairs in the central octanucleotide core in the Pribnow 12-mer duplex with the duplex to strand transition occurring at 55 degrees C in 0.1 M phosphate solution. The magnitude of the NOE measurements between minor groove H-2 protons of adjacent adenosines demonstrates that the base pairs are propeller twisted with the same handedness as observed in the crystalline state. The thymidine imino proton hydrogen exchange at the dA X dT base pairs has been measured from saturation recovery measurements as a function of temperature. The exchange rates and activation barriers show small variations among the four different dA X dT base pairs in the Pribnow 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural studies of the O6meG.C interaction in the d(C-G-C-G-A-A-T-T-C-O6meG-C-G) duplex

One- and two-dimensional nuclear magnetic resonance (NMR) experiments have been undertaken to investigate the conformation of the d(C1-G2-C3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) self-complementary dodecanucleotide (henceforth called O6meG.C 12-mer), which contains C3.O6meG10 interactions in the interior of the helix. We observe intact base pairs at G2.C11 and G4.C9 on either side of the modification site at low temperature though these base pairs are kinetically destabilized in the O6meG.C 12-mer duplex compared to the G.C 12-mer duplex. One-dimensional nuclear Overhauser effects (NOEs) on the exchangeable imino protons demonstrate that the C3 and O6meG10 bases are stacked into the helix and act as spacers between the flanking G2.C11 and G4.C9 base pairs. The nonexchangeable base and H1', H2', H2', H3', and H4' protons have been completely assigned in the O6meG.C 12-mer duplex at 25 degrees C by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) experiments. The observed NOEs and their directionality demonstrate that the O6meG.C 12-mer is a right-handed helix in which the O6meG10 and C3 bases maintain their anti conformation about the glycosidic bond at the modification site. The NOEs between the H8 of O6meG10 and the sugar protons of O6meG10 and adjacent C9 exhibit an altered pattern indicative of a small conformational change from a regular duplex in the C9-O6meG10 step of the O6meG.C 12-mer duplex. We propose a pairing scheme for the C3.O6meG10 interaction at the modification site. Three phosphorus resonances are shifted to low field of the normal spectral dispersion in the O6meG.C 12-mer phosphorus spectrum at low temperature, indicative of an altered phosphodiester backbone at the modification site. These NMR results are compared with the corresponding parameters in the G.C 12-mer, which contains Watson-Crick base pairs at the same position in the helix.     

The conformation of the d(ACCCGGGT) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)2 have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D2O at 12 degrees C. The observed NOE's between the base protons and their own H2' protons and between the base protons and the H2' protons of the 5' adjacent nucleotide and the observed coupling constants between the deoxyribose 1' and 2',2' protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.     

NMR studies of tris-intercalation: solution structure and interaction of d(CTTCGCGCGAAG) with an acridine trimer

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of 1H and 31P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H5', H5"), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O1'endo range, those of the internal guanosines being closer to C2'endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the 3T4C5G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3'-5')G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.     

The Conformation of the d(ACCCGGGT) Duplex in Aqueous Solution

Abstract

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)₂ have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D₂O at 12°C. The observed NOE's between the base protons and their own H2′ protons and between the base protons and the H2′ protons of the 5′adjacent nucleotide and the observed coupling constants between the deoxyribose 1′ and 2′,2″ protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.     

Covalent carcinogenic O6-methylguanosine lesions in DNA. Structural studies of the O6 meG X A and O6meG X G interactions in dodecanucleotide duplexes

High-resolution proton and phosphorus nuclear magnetic resonance studies are reported on the self-complementary d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6meG X A 12-mer when N3 = A3 and O6meG X G 12-mer when N3 = G3), which contain symmetry-related A3 X O6meG10 and G3 X O6meG10 interactions in the interior of the helices. We observe inter-base-pair nuclear Overhauser effects (NOE) between the base protons at the N3 X O6meG10 modification site and protons of flanking G2 X C11 and G4 X C9 base-pairs, indicative of the stacking of N3 and O6meG10 bases in both O6meG X A 12-mer and O6meG X G 12-mer duplexes. We have assigned all the base and a majority of the sugar protons from two-dimensional proton-correlated and nuclear Overhauser effect experiments on the O6meG X A 12-mer duplex and O6meG X G 12-mer duplex in solution. The observed NOEs establish that the A3 and O6meG10 at the modification site and all other residues adopt the anti configuration about the glycosidic bond, and that the O6meG X A 12-mer forms a right-handed duplex. The interaction between the bulky purine A3 and O6meG10 residues in the anti orientation results in large proton chemical shift perturbations at the (G2-A3-G4) X (C9-O6meG10-C11) segments of the helix. By contrast, we demonstrate that the O6meG10 residue adopts a syn configuration, while all other bases adopt an anti configuration about the glycosidic bond in the right-handed O6meG X G 12-mer duplex. This results in altered NOE patterns between the base protons of O6meG10 and the base and sugar protons of flanking C9 and C11 residues in the O6meG X G 12-mer duplex. The phosphorus backbone is perturbed at the modification site in both duplexes, since the phosphorus resonances are dispersed over 2 parts per million in the O6meG X A 12-mer and over 1 part per million in the O6meG X G 12-mer compared to a 0.5 part per million dispersion for an unperturbed DNA helix. We propose tentative pairing schemes for the A3 X O6meG10 and G3 X O6meG10 interactions in the above dodecanucleotide duplexes.     

Sequence-specific recognition of DNA. Nuclear magnetic resonance assignments and structural comparison of wild-type and mutant lambda OR3 operator DNA

The resonances of all the base protons and most of the sugar protons in both strands of the 17 base-pair OR3 operator of the phage lambda, and of the vC3 single base-pair mutant, have been assigned using two-dimensional nuclear magnetic resonance methods. The chemical shift and nuclear Overhauser effect data for these two DNA sequences reveal no structural perturbation at sites distal to the mutation, neither are there significant changes in structure immediately surrounding the altered base-pair in the mutant sequence. These results are consistent with the model proposed by Ohlendorf et al. (1982), based on crystallographic data on the cro protein, for the OR3-cro protein interaction. The data from these solution studies are examined and discussed in the light of this model. This work demonstrates that nuclear magnetic resonance chemical shifts and nuclear Overhauser effect intensities provide a method for comparing the solution structures of DNA molecules. From the resolution available in the spectra of the 17 base-pair operators studied, it is clear that DNA duplexes of up to 30 or more base-pairs can be studied using phase-sensitive methods.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

Sequence-specific recognition of DNA: NMR studies of the imino protons of a synthetic RNA polymerase promoter

We have synthesized both strands of a DNA duplex containing the consensus Pribnow promoter sequence TATAATG , flanked by GC base pairs to stabilize the ends of the helix. The stability of this duplex has been studied by using 1H nuclear magnetic resonance. The imino protons have been assigned by using the sequential nuclear Overhauser effect approach. Exchange rates have been monitored by using selective inversion recovery measurements. The helix is relatively unstable in the center of the AT-rich region even when surrounded by GC base pairs, and there is considerable asymmetry in the melting of the helix.     

NMR Studies of Tris-Intercalation: Solution Structure and Interaction of d(CTTCGCGCGAAG) with an Acridine Trimer

Abstract

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of ¹H and ³¹P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H₅', H_5”), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O_1′ endo range, those of the internal guanosines being closer to C_2′endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the ³T⁴C⁵G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3′-5′)G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.     

Two dimensional NMR studies on the solution structure of d-CTCGAGCTCGAG

Two dimensional (2D) FT-NMR investigations have been carried out on the self-complementary dodecanucleotide d-CTCGAGCTCGAG, which has cleavage sites for the restriction enzyme Xho I (between C and T). The central TCG portion is also known to show a preference for DNAase activity. Complete resonance assignments have been obtained for the non-exchangeable sugar and base protons of the oligonucleotide. Information regarding sugar geometries, glycosidic torsion angles and other structural parameters has been obtained from the relative intensities of the cross peaks in the COSY and NOESY spectra. The results indicate that deoxyribose rings of C1 and C7 adopt a conformation different from the remaining sugars in the double helical oligonucleotide. The central TCG portion also exhibits variations in the backbone structure. The base stacking in the double helix shows interesting sequence dependent effects suggesting that the sequence effects are not localised to nearest neighbours but extended over longer stretches.     

Sequence-dependent recognition of DNA duplexes: netropsin complexation to the TATA site of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

We have recorded one-dimensional exchangeable proton and two-dimensional nonexchangeable proton nmr spectra on the complex of netropsin with the self-complementary d(G-G-T-A-T-A-C-C) duplex in aqueous solution between 25° and 35°C. The antibiotic amide, pyrrole, and methylene protons, and the nucleic acid base and sugar H1′, H2′, H2″, and H3′ protons, have been assigned from an analysis of the two-dimensional nuclear Overhauser effect (NOESY) spectra of the complex. We observe intermolecular NOEs between the antibiotic concave face amide, pyrrole, and CH₂ resonances, and the adenosine H2 and sugar H1′ protons of base-pairs T3·A6 and A4·T5 in the central TATA core of the d(G₁-G₂-T₃-A₄-T₅-A₆-C₇-C₈) duplex. We present a molecular model outlining these seven antibiotic-DNA contacts for the complex in solution. The observed line-broadening of several base and sugar protons at the TATA minor groove netropsin binding site in the complex at 35°C are interpreted in terms of intermediate exchange between two orientations of bound netropsin on the duplex.     

Nuclear overhauser effect study of yeast aspartate transfer ribonucleic acid

Nuclear Overhauser effect studies are described for yeast tRNAAsp in 0.1 M NaCl, pH 7.0. A primary aim is to develop a general method for attacking the problem of assignment in transfer ribonucleic acids (tRNAs). Previously, we have demonstrated the utility of the nuclear Overhauser effect (NOE) between protons on adjacent base pairs combined with C8 deuterium substitution, by assigning the imino protons of the dihydrouridine stem and the two reverse-Hoogsteen base pairs T54-A58 and U8-A14. Here, we extend that approach to other parts of the molecule. We also describe several NOE-connected patterns for, e:g., m5CG and psi 55 N3H imino protons which may be of general utility. For the first time, a purine-15-pyrimidine-48 base pair (in this case A15-U48) has been assigned. A total of 13 of 25 base pairs from all parts of the molecule and several noninternally bonded imino protons have now been assigned unambiguously. This is a general method for assigning resonances in tRNA and perhaps in all double-stranded nucleic acids. This, and the distance information inherent in NOE measurements, should make NMR more generally applicable to nucleic acids.