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The thermodynamics of oligonucleotide-directed triple helix formation at single DNA sites

Citation

Singleton, Scott F. (1995) The thermodynamics of oligonucleotide-directed triple helix formation at single DNA sites. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10242007-090557

Abstract

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The local triple-helical complexes formed upon binding of pyrimidine oligonucleotides to double-helical DNA offer a versatile structural motif for designing sequence specific duplex DNA-binding molecules. The experimental determination of oligonucleotide association constants as a function of solution conditions is necessary to characterize the noncovalent forces which contribute to the affinity and specificity of DNA recognition by triple helix formation. Such information will be important for the rational application of oligonucleotide-directed triple helix formation to such tasks as the manipulation of large DNAs, and the artificial modulation of biological events. We are interested in controlling the relative amount of a local triple-helical complex formed at equilibrium under conditions which approximate those found within cells. Thus, we chose to measure equilibrium constants for the formation of triple-helical complexes at single sites within larger duplex DNAs in mixed valence salt solutions near neutral pH.

Concepts borrowed from existing footprint titration methods were combined with the powerful affinity cleaving strategy in the development of the affinity cleavage titration method for the measurement of single-site DNA-binding isotherms (Chapters One and Two). The amount of site-specific cleavage of a radiolabeled plasmid DNA fragment produced by a 15mer oligonucleotide-EDTA[...]Fe is measured over an oligonucleotide concentration range covering four orders of magnitude, allowing a binding curve to be constructed. Curve-fitting a Langmuir titration isotherm to the data points affords an equilibrium binding constant, [...], that is identical within experimental uncertainty to those obtained from quantitative DNase I footprint titrations of the same oligonucleotide with and without EDTA[...]In. Further affinity cleavage titrations demonstrate that the energetic stabilization of this local triple-helical structure depends on the length of the oligonucleotide, the presence of base triplet mismatches, and the solution conditions, including the pH, the concentration and valence of cations, and the temperature.

Decreasing the length of the oligonucleotide from 15 to 11 nts reduces the stability of the corresponding triplexes by ~0.3 kcal/mol per nucleotide removed (Chapter Two). Single internal base triplet mismatches result in a destabilization of the local triple-helical structure by ~ 3 kcal/mol. The equilibrium association constants for the 15mer increased by 10-fold as the pH was decreased from pH 7.6 to pH 5.8, indicating that the corresponding triple-helical complex was stabilized by 1.4 kcal[...]mol[superscript -1] at the lower pH (Chapter Three). Equilibrium association constants for the 15mer in the presence of various concentrations of KCl,MgCl[subscript 2], and spermine tetrahydrochloride (SpmCl[subscript 4]) are reported in Chapter Four. Varying the K[...] concentration by a factor of 28 in the presence NaCl, MgCl[subscript 2], and SpmCl[subscript 4] resulted in an overall 100-fold decrease in the binding affinity from the lowest to the highest concentration. In contrast, measured binding constants increased 500-fold as the Spm[superscript 4+] concentration was increased 10-fold.

There was a modest effect on the binding constant (a 3-fold decrease) upon increasing the Mg[superscript 2+] concentration by a factor of 100. The influence of temperature on the energetics of oligonucleotide-directed triple helix formation in mixed valence salt solutions at pH 7.0 are reported in Chapter Five. In three solutions differing in their salt compositions, the equilibrium association constants decreased at least 100-fold (from >10[superscript 7]M[superscript -1] to ~ 10[superscript 5]M[superscript -1]) as the temperature was increased from 8°C to 37°C. Least squares analysis of van't Hoff plots (lnK versus 1/T) of the data revealed that in each solution the triplex is enthalpically stabilized by ~ 2 kcal per mole of base triplets.

This value for the enthalpic contribution to triplex stability, which reflects contributions from both T[...]AT and C+GC base triplets, is identical within experimental error to that measured previously using differential scanning calorimetry for the 15mer binding to 15 by within a 21-bp duplex. We collaborated with Professor Kenneth J. Breslauer of Rutgers University to make the calorimetric measurement, and the complete thermodynamic characterization of the stability and the melting behavior of the triplex are described in Chapter Six.

Taken together, the equilibrium association constants, the thermal denaturation data, and the calorimetric data reported here provide a quantitative measure of the influence of third strand length, base triplet mismatches, pH, salt, and temperature on the stability and the melting behavior of a DNA triplex. Such information should prove useful in designing triplex-forming oligonucleotides and in defining solution conditions for the effective use of triple-helical structure formation as a tool for modulating biochemical events.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Unknown, Unknown
Defense Date:24 June 1994
Record Number:CaltechETD:etd-10242007-090557
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-10242007-090557
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4237
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:24 Oct 2007
Last Modified:26 Dec 2012 03:06

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