Conformational analysis of the octamer d(G‐G‐C‐C‐G‐G‐C‐C) in aqueous solution

Abstract

Proton NMR studies in H 2 0 and 'HzO 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, 3333-33461. Base, Hl', 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.Recent advances in DNA synthesis have opened the possibility of studying oligonucleotides of known specific sequence. So far, X-ray crystallography and high-field NMR spectroscopy have proved powerful tools in the study of the crystalline and solution structure of oligonucleotides, respectively. 2D NMR techniques have opened the way to the study of long DNA fragments. 2D correlation (COSY) experiments have been found useful to establish scalar connectivities between protons. In this way sets of proton resonances can be distinguished, each set belonging to a different sugar residue. In addition, 2D NOE (NOESY) experiments allow one to find intra-and inter-residual connectivities between base and sugar protons and thus to assign many of the resonances in the proton spectra [l -171. NOESY signal intensities serve to determine the helical type [3, 151 and local structural features of oligonucleotide fragments [5, 7, 18, 191 and, finally, to find answers to questions concerning possible differences between Correspondence to C . Altona, Gorlaeus Laboratoria der Rijksuniversiteit te Leiden,

Influence of the base sequence on the conformational behaviour of DNA polynucleotides in solution

“…three-, four-or multistate) temperature-dependent conformational transition. Quite similar observations have been reported for the base proton and H1' resonances of the 3'-terminal residues dC(8) [36] and of residue dC(4) of d(G-G-C-C)2 [42], as well as for the base proton resonances of residues dC (8) [44]. Taken together, these observations strongly suggest that the bases at a 3'-terminus consisting of two or more pyrimidine residues upon cooling are only gradually engaged into a regular stacking interaction with their 5' neighbour when the remaining residues are already regularly stacked in the intact duplex [36].…”

“…Quite similar observations have been reported for the base proton and H1' resonances of the 3'-terminal residues dC(8) [36] and of residue dC(4) of d(G-G-C-C)2 [42], as well as for the base proton resonances of residues dC (8) [44]. Taken together, these observations strongly suggest that the bases at a 3'-terminus consisting of two or more pyrimidine residues upon cooling are only gradually engaged into a regular stacking interaction with their 5' neighbour when the remaining residues are already regularly stacked in the intact duplex [36]. In other words, upon warming up, 'early melting' of the 3'-terminal dC residue occurs when the 3'-terminal consists of the sequence -C-C, whereas two residues appear to be involved in such behaviour when the DNA is terminated by -C-T-C. At this point it should be stressed that: (a) 3'-terminal DNA sequences of the type -R-R, -R-Y or -Y-R appear to display shift profiles like those of the core residues, see e.g.…”

“…The chemical shift values observed at 300 K are collected in Table 2. Discrimination between the H2' and H 2 resonances followed from protonproton coupling constant considerations (see below and [36]). Note that the H2' resonances of all residues in the intact duplex of the two octamers, except for the 3'-terminal residues, occur upfield relative to the corresponding H2" resonance, in accordance with a rule proposed earlier by us [36].…”

“…Discrimination between the H2' and H 2 resonances followed from protonproton coupling constant considerations (see below and [36]). Note that the H2' resonances of all residues in the intact duplex of the two octamers, except for the 3'-terminal residues, occur upfield relative to the corresponding H2" resonance, in accordance with a rule proposed earlier by us [36]. The observation of relatively strong intraresidue base(n)-H2'(n) NOEs in combination with relatively strong interresidue base(n)-H2"(n-l) NOEs [36, 371 indicates that the compounds in the present study adopt predominantly a B-DNA-type helix in solution; see also the section concerned with the conformation of the sugar ring.…”

“…K-'). A flat low-temperature basis line was assumed [36] ; the temperature-dependent base line above about 350 K is taken into account in the DUPSTAK model. Table 4 shows the thermodynamic parameters obtained by taking the weighted average of the proton analyses per residue; no parameters could be deduced for residues dT(7) and dC (8) The thermodynamic parameters for duplex formation determined for d(C-A-C-A-T-G-T-G)z compare well with those predicted from the base sequence using thermodynamic data derived from optical melting studies on long DNA fragments [7] (Table 4).…”