SUNY at Albany
June 19-23, 2001
High-resolution NMR Structure of an AT-rich DNA Fragment Refined with the miniCarlo Program
AT-rich DNA sequence motifs are heavily involved in regulation of DNA transcription and regulation; structural analysis of such sequences is important for better understanding of these processes. However, the low thermal stability of AT-rich oligonucleotides is a significant obstacle to determining their high-resolution structures in solution. We have largely overcome this problem by placing AT-rich sequences in stem-and-loop constructs with the "extraordinarily stable" DNA hairpin loop GAA (1). Here, we describe proton NMR studies and NOE-based determination of a high-resolution structure for a monomolecular DNA construct which includes nine contiguous AT base pairs:5'-C-C-T-A-A-T-T-A-T-A-A-C-G
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At physiological solution conditions, double-stranded DNA segments always adopt a B-type conformation, with a standard (sequence-independent) pattern of nuclear Overhauser effect (NOE) cross peaks. Determination of high-resolution sequence-dependent three-dimensional structure based on NOE data requires, therefore, careful quantification of NOE volumes and rigorous determination of interproton distances. For this purpose, we use a full relaxation matrix algorithm (Mardigras) with an error analysis procedure (2). The calculated distance bounds are then used with the miniCarlo program which models nucleic acids based on a set of internal coordinates -- helical parameters (3). The structure is refined with miniCarlo using a Metropolis Monte Carlo simulated annealing protocol (4) starting with a set of Dyana-generated (5) random conformations. The refined structure displays a number of sequence-dependent features, such as characteristic stacking patterns of ApA:TpT, ApT:ApT and TpA:TpA base pair steps, and sequence-dependent variation of the minor groove width.
Nikolai B. Ulyanov (1), William R. Bauer (2) and Thomas L. James (1*)
Department of Pharmaceutical Chemistry(1), University of California at San Francisco, CA 94143-0446; Department of Molecular Genetics and Microbiology(2), Health Science Center, SUNY at Stony Brook, Stony Brook, NY 11794-5222