Energetics of Triple Helix Formation by Oligonucleotides Containing Nonnatural Bases

Author: Priestley, Eldon Scott

Year: 1996

Degree: Dissertation (Ph.D.)

Advisors: Goldreich, Peter Martin; Dervan, Peter B.

Committee Members: Myers, Andrew G.; Rees, Douglas C.; Imperiali, Barbara; Dervan, Peter B.

Option: Chemistry

DOI: 10.7907/ja6x-c417

Abstract

Oligonucleotide directed triple helix formation is a powerful chemical approach to the sequence specific recognition of duplex DNA. In this approach, pyrimidine oligonucleotides bind homopurine duplex sequences by formation of T·AT and C+GC base triplets. Likewise, purine oligonucleotides also bind homopurine sequences by formation of G·GC and either A·AT or T·AT triplets. The two motifs differ in that the third strand oligonucleotide binds parallel to the homopurine duplex strand in the "pyrimidine motif" and antiparallel in the "purine motif." This approach currently lacks generality, since there are no third strand bases available for highly specific, high affinity recognition of CG and TA base pairs in either structural motif. In addition, the pyrimidine motif suffers from pH dependent triplex stability introduced by the C+GC base triplets, while the purine motif suffers from competing aggregation due to G quartet formation by the G-rich third strand. In principle, these problems may be addressed by the development of nonnatural bases with the desired properties.

This thesis describes the application of quantitative DNase footprinting methods to determine the energetics of triple helix formation by oligonucleotides containing nonnatural bases. Chapter One provides an introduction to current research on triple helix formation. Chapter Two describes an NMR study of an intramolecular triplex containing the nonnatural base P, which provides pH independent recognition of GC base pairs. Chapter Three describes the quantitation of the specificity and sequence composition effects for triple helix formation by oligonucleotides containing P, using DNase footprinting. The specificity assay has proven particularly useful for initial characterization of a variety of novel bases. In Chapter Four, the synthesis and binding studies of the desmethyl derivative of P are reported. The results indicate that removal of the methyl group has a small detrimental effect on triple helix formation. Chapter Five describes an investigation of the specificity of the novel base 2-pyrimidinone. Chapter Six introduces the nonnatural base N7-deoxyguanosine, which is shown to be a replacement for P as a base for pH independent GC recognition, since it shows slightly improved affinity and is synthetically more accessible. Chapter Seven describes attempts to develop a new structural motif for triple helix formation based on N7-substituted purine nucleosides.

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