Fine structure of nucleic acid hairpins and duplexes

Stephen Michael Testa, Purdue University

Abstract

Studies of the relationship between nucleic acid structure and function are limited by the rudimentary understanding of the forces that drive structure formation. New methods for identifying the structure and stability of nucleic acid fragments have been developed, and these are expected to contribute to the understanding of nucleic acid structure formation. In this work electronic transitions of the n$\pi\sp\*$ type, produced when azine nitrogens are exposed to long wavelength ultraviolet energy, have been exploited for the analysis of the state of denaturation and the global geometry of nucleic acid helices. The interaction of these collinearly oriented transition dipoles with one another results in an increase in the absorbance of structured oligonucleotides at wavelengths above 280 nm. Thermal denaturation of these helices at 296 nm results in a decrease in their absorbance. This hypochromic effect has been used to define the stability and molecularity of nucleic acid structures. Since the extent of collinearity of the azine n$\pi\sp\*$ transition dipoles in B-DNA is different from that in Z-DNA, long wavelength percent hypochromicity has been used to identify and study these helical conformations. The biological relevance of oligonucleotides that assume Z-helical conformations has been uncertain because of the requirement for salt concentrations at least ten times higher than that found in vivo. This work circumstantiates that, although most Z-helices require very high salt conditions for Z-helix formation, certain hairpins assume Z-helical conformations in near physiological salt conditions. One example is the tridecamer CGCGAGTGACGCG. Long wavelength thermal denaturation analyses of specific variants of this tridecamer have revealed that the formation of a Z-helix in low salt by the CGCG stem of this hairpin is induced by the loop region. The sequence requirements of these Z-helix inducing hairpin loops are adenines immediately adjacent to the top of the stem, purines (with at least one guanine) in the loop positions adjacent to the adenines, and a thymine in the central loop position. From these studies it is expected that Z-helical hairpins exist in vivo within single-stranded regions of DNA.

Degree

Ph.D.

Advisors

Gilham, Purdue University.

Subject Area

Biology|Biophysics|Biochemistry

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