Duplex In Aqueous Solution Research Articles

Conformation of Adenosine Bulge-containing Deoxytridecanucleotide Duplexes in Solution: Extra adenosine stacks into duplex independent of flanking sequence and temperature

Structural features at the extra adenosine bulge sites in DNA duplexes have been elucidated from an NMR analysis of two-dimensional through space and through bond connectivities in the self-complementary d(C-C-G-G-A-A-T-T-C-A-C-G-G) (CAC 13-mer) and d(C-C-G-A-G-A-A-T-T-C-C-G-G) (GAG 13-mer) duplexes in aqueous solution. These studies establish that the extra adenosine stacks into the helix at all temperatures below the onset of the melting transition in solution, and the results are independent of whether the extra adenosine is flanked by cytidines (CAC 13-mer) or guanosines (GAG 13-mer). The NMR parameters establish that the extra adenosine can be accommodated into the helix with the flanking base pairs adopting a wedge-shaped orientation. The resulting perturbation extends out to the C10-G11 phosphodiester backbone adjacent to the bulge segment in both the CAC 13-mer and GAG 13-mer duplexes.

NMR studies of echinomycin bisintercalation complexes with d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution: sequence-dependent formation of Hoogsteen A1.T4 and Watson--Crick T1.A4 base pairs flanking the bisintercalation site.

We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Palindromic octa- and dodecanucleotides containing 2'-deoxytubercidin: synthesis, hairpin formation, and recognition by the endodeoxyribonuclease EcoRI.

Octa- and dodecanucleotides containing 2'-deoxytubericidin within the endodeoxyribonuclease EcoRI recognition fragment d(GAATTC) have been prepared by solid-phase synthesis. Whereas octamers as well as dodecamers with a "random" flanking region formed duplexes in aqueous solution, the dodecamer d(CGCGAATTCGCG) and isosterically modified oligomers thereof showed a strong tendency of hairpin formation. Due to this, cleavage with the endodeoxyribonuclease EcoRI was strongly decreased. In contrast, d(GTAGAATTCTAC) was easily cleaved by the enzyme. Single replacement of one of the dA residues by 2'-deoxytubercidin within the recognition sequence decreased the cleavage velocity but retained specificity. Twofold modification prevents cleavage of the oligomer. This implies that both N-7 purine nitrogens are proton acceptor sites for the endodeoxyribonuclease EcoRI.

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.

Conformational analysis of the octamer d(G-G-C-C-G-G-C-C) in aqueous solution. A one-dimensional and two-dimensional proton NMR study at 300 MHz and 500 MHz.

Proton NMR studies in H2O and 2H2O were carried out on the self-complementary DNA octamer d(G-G-C-C-G-G-C-C) and compared with similar studies on the dimethylated analogue d(G-G-m5C-m5C-G-G-C-C) [Sanderson, M. R., Mellema, J.-R., van der Marel, G. A., Wille, G., van Boom, J. H. & Altona, C. (1983) Nucleic Acids Res. 11, 3333-3346]. Base, H1', H2' and H2" resonances were assigned by means of two-dimensional nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments. Chemical shift vs temperature profiles were used to analyze the temperature-dependent conformational behaviour and to extract thermodynamic parameters for the duplex-to-coil transition. Analysis of proton-proton couplings were used to discriminate between J1'2' and J1'2" and between the H2' and H2" resonances as well as to obtain conformational parameters of the sugar rings. From the NOESY and COSY experiments, the temperature profiles and the analysis of the coupling data it is concluded that: (a) the compound adopts a B-DNA-type helix in solution; (b) the sugar rings in the intact duplex display limited conformational freedom; (c) methylation of the cytosine bases at the C5 position has no measurable effect on the conformational behaviour of the octamer, nor on the conformation of the sugar rings; however, methylation does increase the stability of the duplex in aqueous solution under conditions of low salt concentration.

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.

AbstractWe 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.

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

AbstractThe 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.

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-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.

Use of inter-proton nuclear Overhauser effects to assign the nuclear magnetic resonance spectra of oligodeoxynucleotide and hybrid duplexes in aqueous solution.

Inter-proton nuclear Overhauser enhancements (NOEs) have been used to assign the aromatic, anomeric and 2' resonances in the 1H nuclear magnetic resonance spectrum of the duplex formed between d(T-C-A-C-A-T) and d(A-T-G-T-G-A). The same techniques have been applied to assignments in the hybrid duplex formed by d(T-C-A-C-A-T) with r(A-U-G-U-G-A). Comparison of intra-residue with inter-residue NOEs yields structural information which suggests that the conformations of both duplexes are similar. The NOEs are consistent with inter-proton distances measured from models of B-form DNA. Circular dichroic results confirm these deductions.

Proton nuclear Overhauser effect study of the structure of a deoxyoligonucleotide duplex in aqueous solution.

Nuclear Overhauser enhancements (NOEs) have been observed between some of the nonexchangeable protons of the self-complementary octanucleoside heptaphosphate d(G-G-T-A-T-A-C-C) in conditions under which duplex formation takes place. Comparison of inter- and intraresidue NOEs has made possible the assignment of all the aromatic and 1'- and 2'-ribosyl signals. In particular, and with the exception of the 5' terminal, NOEs of comparable magnitude are observed between H-8 of purines or H-6 of pyrimidines and pairs of anomeric protons. There are the H-1's of the nucleotide containing the relevant aromatic proton and those of the adjacent residue in the 5' direction. Differentially evolving NOEs are also generated between aromatic protons and the C-2' methylene protons on both the same residue and on the neighboring 5' residue. The relative magnitudes of these NOEs are discussed in terms of overall helical geometry and are consistent with a generally accepted B-type DNA model (R. Chandrasekaran and S. Arnott, personal communications).

Proton NMR studies on the covalently linked RNA-DNA hybrid r(GCG)d(TATACGC). Assignment of proton resonances by application of the nuclear Overhauser effect.

Proton NMR spectra of a covalently linked self-complementary RNA X DNA hybrid, r(GCG)-d(TATACGC), are recorded in H2O and D2O. Imino proton resonances as well as the non-exchangeable base and H-1' resonances are unambiguously assigned by means of nuclear. Overhauser effect measurements. Additional information was obtained by 31P NMR and circular dichroism spectra. The RNA parts in the duplex attain full conformational purity and adopt the usual A-RNA conformation. The DNA residues opposite the RNA tract do not adopt an A-type structure completely. Their respective sugar rings still appear to possess a certain conformational freedom. The same holds true for the central d(-TATA-) sequence which forms a DNA X DNA duplex. There appears to be a structural break in this part: the first two residues, T(4) and A(5), are clearly influenced by the adjacent RNA structure, whereas residues T(6) and A(7) behave quite similar to what usually is found in DNA duplexes in aqueous solution.

Mutual interaction between adjacent dG . dC actinomycin binding sites and dA . dT netropsin binding sites on the self-complementary d(C-G-C-G-A-A-T-T-C-G-C-G) duplex in solution.

The Watson-Crick imino protons, the backbone phosphodiester resonances, and the antibiotic exchangeable protons have been used as markers to monitor the separate and simultaneous binding of actinomycin and netropsin to the d(C-G-C-G-A-A-T-T-C-G-C-G) self-complementary duplex in aqueous solution. We demonstrate that intercalation of actinomycin at dG(3'-5')dC sites at either end of the duplex results in a conformational perturbation at the dA . dT tetranucleotide core of the dodecanucleotide duplex. Parallel studies of the groove binding of netropsin at dA . dT sites in the interior of the duplex reveal a conformational perturbation which extends to adjacent dG . dC base pairs in the dodecanucleotide duplex. The NMR markers demonstrate that the d(C-G-C-G-A-A-T-T-C-G-C-G) duplex can accommodate actinomycin and netropsin simultaneously at adjacent dG . dC and dA . dT tetranucleotide blocks along its length with some mutual interaction between neighboring antibiotic binding sites.