DNA POLYMORPHISM IN SOLUTION--A THERMODYNAMIC STUDY

Project Details

Description

We propose to continue our thermodynamic characterizations of the molecular
forces that control the stability and the conformational preferences of
nucleic acid molecules in solution. Our ultimate objective is to establish
a comprehensive thermodynamic library that provides the data base needed to
evaluate sequence-specific, structure-specific, and solvent-specific
conformational preferences of functionally-important domains within
naturally-occurring nucleic acids. The thermodynamic data required to achieve these goals will be obtained by
applying the techniques of microcalorimetry (both isothermal mixing and
temperature scanning) to characterize helix forming and helix disrupting
events as well as helix-to-helix conformational transitions in specially
designed and synthesized oligomeric and polymeric nucleic acid molecules
which possess sequences that will be systematically varied. This approach
has allowed us to correlate measured thermodynamic parameters with specific
structural and/or conformational features defined by uv and CD spectroscopy
as well as by high field NMR. In fact, during the previous budget period,
we used this combination of spectroscopic and calorimetric techniques to
characterize thermodynamically all ten nearest-neighbor Waston-Crick
interactions as well as a variety of DNA secondary structural forms of
biological interest (e.g. hairpins, duplexes with dangling ends, duplexes
with abasic sites, immobile junctions, "dumbbells," etc.). During the next
budget period, as described below, we will focus our calorimetric studies
on additional nucleic acid nucleic acid structures of biological
significate which have yet to be thermodynamically characterized. To be specific, during the requested budget period we propose to determine
as a function of base sequence, base modification, and solution conditions
the relative stabilities (DeltaGo), the temperature-dependent transitions
(DeltaHo, DeltaCp), and the melting cooperativities (DeltaHv.H/DeltaHcal)
of the following nucleic acid systems: DNA duplexes with base modified
mutagenic lesions (e.g. exocyclic and alkylated adducts); DNA triplexes;
DNA duplexes with distortions which when properly phased give rise to
"bending"; DNA hairpins; DNA dumbbell-shaped structures; DNA duplexes with
dangling ends; and DNA/RNA hybrid duplexes. The thermodynamic data we
obtain from these proposed studies will substantially expand our existing
library, thereby providing us with a broadened and improved empirical basis
for evaluating the relative stabilities and structure, and solution
conditions. The thermodynamic data also will assist us in evaluating the
degree to which sequence-, structure-, and solvent-induced conformational
distortions nad transformations contribute to the overall driving forces of
biologically significant processes. Ultimately, we hope to establish a
phase diagram for DNA (including DNA/RNA hybrid duplexes) in which we
define the relative stabilities and map the temperature- and solvent-
induced interconversions of sequence-specific conformational states. Considering the potential roles of base modification and/or conformational
heterogeneity in mechanisms for selective, local control of events such as
protein-nucleic acid interactions, drug-DNA binding, gene expression, and
DNA packaging, an ability to predict sequence-dependent, local
conformational preferences and transformations in DNA and in DNA/RNA
polymers is of the utmost importance. The calorimetric experiments
described in this proposal ar designed to provide the thermodynamic data
required to establish this predictive ability so that sequences favoring
specific structural forms can be identified and correlated with particular
functional roles.
StatusFinished
Effective start/end date4/1/793/31/00

Funding

  • National Institutes of Health: $214,738.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $231,838.00
  • National Institutes of Health
  • National Institutes of Health: $272,860.00
  • National Institutes of Health: $183,522.00
  • National Institutes of Health: $292,839.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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