Oligonucleotide-Directed Alkylation of Double Stranded DNA: a Product and Kinetic Analysis

Author: Taylor, Matthew Jabez

Year: 1996

Degree: Dissertation (Ph.D.)

Advisor: Goldreich, Peter Martin

Committee Members: Rees, Douglas C.; Dervan, Peter B.; Dougherty, Dennis A.; Myers, Andrew G.; Goldreich, Peter Martin

Option: Chemistry

DOI: 10.7907/61av-hm22

Abstract

This thesis research focuses on providing a better understanding of a methodology developed in the group of Professor Peter B. Dervan for the sequence-specific alkylation of double stranded DNA. It was previously reported from this group that an oligonucleotide equipped with an electrophilic bromoacetyl moiety could be used to cleave yeast chromosome III (340 kilobase pairs) at a single site in high yield. Attachment of the nondiffusible electrophile to the 5-position of a thymine at the 5'-end of a pyrimidine oligodeoxyribonucleotide affords sequence-specific alkylation of a guanine two base pairs to the 5'-side of a local triple-helical complex. Warming and base treatment then produces cleavage at this site. We have identified the products of this reaction. Consistent with an alkylation / depurination mechanism is the formation of phosphate termini in the oligonucleotide products, and identification of the oligonucleotide-guanine conjugate produced in the reaction was achieved, demonstrating that this reaction for cleavage of double helical DNA is atom-specific for the N7-position of guanine in duplex DNA.

Limitations in specificity for these systems will be due, in part, to the rate enhancement achieved upon covalent attachment of the electrophile to the DNA-binding ligand. We now understand this in quantitative terms following a comparison of the rate of bimolecular reaction of 2-bromoacetamide at the N7-position of guanine in duplex DNA to the rate of unimolecular reaction at N7 of guanine mediated by triple helix formation. Employing a polyacrylamide gel assay, these rate constants were determined and provide data indicating a maximum rate enhancement upon localization of bromoacetyl by triple helix formation of ten-million fold. The work described here has given us a detailed understanding of this system, which couples sequence-specific recognition one million times more specific than restriction enzymes with atom-specific reactivity ten million times faster than its bimolecular counterpart, and represents a foundation for development of future systems of this class.

Part two of this thesis describes our efforts to extend oligonucleotide-directed alkylation to double stranded RNA. Because it has been shown that only RNA single strands bind double-helical RNA by triple helix formation, an N-bromoacetyloligoribonucleotide was synthesized. Described is the synthesis of the phosphoramidite necessary for the synthesis of this novel RNA-electrophile conjugate. In collaboration with Jean-Francois Mouscadet, a postdoctoral fellow in the Dervan group, it was found that triplex helix mediated alkylation of double stranded RNA occurred with only modest efficiency. It is likely that solutions to this problem will be attained only through long-term efforts beyond the scope of this thesis.

Part three describes our attempts to determine an x-ray crystal structure of a triple-helical complex. In collaboration with Professor Doug Rees at Caltech, various triple-helical complexes of the pyrimidine•purine•pyrimidine type were prepared on a ten micromole scale and crystallography experiments were performed. Single crystals were obtained which diffracted to high resolution, and analysis of these crystals indicated a stoiciometry consisted with triple-helical complexes. No solution of any of the complexes giving x-ray quality crystals were obtained. Documented in this work is methodology for the synthesis and purification of 5-i'odo-2'-deoxyuridine-containing oligonucleotides. At this time, no high resolution x-ray crystal structure of these complexes has been reported, highlighting the continued importance of efforts in this area.

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