Albany 2001

category image Biomolecular
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:

___ | | | | | | | | | | | | A

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.

    References and Footnotes
  1. Hirao I., Kawai G., Yoshizawa S., Nishimura Y., Ishido Y., Watanabe K. & Miura K. Nucl. Acids Res. 22, 576-582, 1994.
  2. Liu H., Spielmann H.P., Ulyanov N.B., Wemmer D.E. & James T.L. J. Biomolec. NMR 6, 390-402, 1995.
  3. Zhurkin V.B., Lysov Yu.P. & Ivanov V.I. Biopolymers 17, 377-412, 1978.
  4. Ulyanov N.B., Schmitz U. & James T.L. J. Biomolec. NMR 3, 547-568, 1993.
  5. Guntert P., Mumenthaler C. & Wuthrich K. J. Mol. Biol. 273, 283-298, 1997.

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
Phone: 415-476-1916; Fax: 415-502-4690; Email: james@picasso.nmr.ucsf.edu