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.

Base Sequence↗

PubMedDate not supplied

Molecular recognition in noncovalent antitumor agent-DNA complexes: NMR studies of the base and sequence dependent recognition of the DNA minor groove by netropsin.

We have investigated intermolecular interactions and conformational features of the netropsin complexes with d(G1-G2-A3-A4-T5-T6-C7-C8) duplex (AATT 8-mer) and the d(G1-G2-T3-A4-T5-A6-C7-C8) duplex (TATA 8-mer) by one and two-dimensional NMR studies in solution. We have assigned the amide, pyrrole and methylene 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. The directionality of the observed distance-dependent NOEs demonstrates that the 8-mer helices remain right-handed and that the arrangement of concave and convex face protons of netropsin are retained in the complexes. The observed changes in NOE patterns and chemical shift changes on complex formation suggest small conformational changes in the nucleic acid at the AATT and TATA antibiotic binding sites and possibly the flanking G.C base pairs. We observe intermolecular NOEs 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.T5 base pairs of the AATT 8-mer and TATA 8-mer duplexes. The concave face pyrrole protons of the antibiotic also exhibit NOEs to the sugar H1' protons of residues 5 and 6 in the AATT and TATA 8-mer complexes. We also detect intermolecular NOEs between the guanidino and propioamidino methylene protons at either end of netropsin and the adenosine H2 proton of the two flanking A3.T6 base pairs in the AATT 8-mer and T3.A6 base pairs in the TATA 8-mer duplexes. These studies establish a set of nine contacts between the concave face of the antibiotic and the minor groove AATT segment and TATA segment of the 8-mer duplexes in solution. The observed magnitude of the NOEs require that there be no intervening water molecules sandwiched between the concave face of the antibiotic and the minor groove of the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation. The observed differences in the netropsin amide proton chemical shifts in the AATT 8-mer and TATA 8-mer complexes suggest differences in the strength and/or type of intermolecular hydrogen bonds at the AATT and TATA binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗

PubMedDate not supplied

Comparison of methods to assess coronary heart disease prevalence in South Asians.

BACKGROUND: Migrants from the Indian subcontinent (South Asian migrants) in the United Kingdom have high mortality from coronary heart disease (CHD) in comparison to the indigenous population. Few studies have assessed the prevalence of CHD in South Asians, and the applicability of conventional survey methods in this population is not known. In this pilot random population survey of South Asian men and women living in West London, the prevalence of CHD as judged by the Rose questionnaire, past cardiac history, cardiologist and resting electrocardiogram were compared. METHODS: Subjects aged 30-64 years from randomly selected households were invited for a cardiological assessment. A lay person administered the Rose questionnaire and recorded the past cardiac history. A cardiologist also made an independent assessment and a 12-lead electrocardiogram was recorded and analysed according to the Minnesota code. RESULTS: Three hundred and seventy-six individuals (192 men and 184 women) were assessed. The prevalence of angina in men and women, respectively, was 3.1% and 4.9% by the Rose questionnaire; 2.6% and 2.2% by past cardiac history; and 4.2% and 0.5% according to the cardiologist. The prevalence of myocardial infarction in men and women, respectively, was 5.2% and 2.2% by the Rose questionnaire, 3.6% and zero by past cardiac history and 3.6% and 0.5% by the cardiologist. Q/QS codes were present in 1.6% men and 0.5% women and ischaemic codes in 13% men and 14% women. Ischaemic changes were not associated with any cardiac history in 72% of men and 92% of women. For a diagnosis of CHD in men, there was poor agreement between the Rose questionnaire and either the past cardiac history or the cardiologist's assessment, but moderate agreement between the past cardiac history and the cardiologist. Agreement was poor between all three methods for a positive diagnosis of CHD in women. CONCLUSION: Current accepted epidemiological methods for assessing CHD prevalence may be inaccurate in South Asians, especially women. Electrocardiogram abnormalities suggestive of ischaemia are common in South Asians and are usually not associated with evidence of CHD. Thus, their value as indicators of CHD is questionable.

Adult↗