Oligonucleotide directed sequence specific recognition and alkylation of double helical DNA by triple helix formation

Citation

Povsic, Thomas J. (1992) Oligonucleotide directed sequence specific recognition and alkylation of double helical DNA by triple helix formation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/krbx-2z47. https://resolver.caltech.edu/CaltechTHESIS:09222011-113738481

Abstract

Chapter II: Footprinting of Oligonucleotides on Double Helical DNA using MPE•Fe(II), DNase I, and Dimethyl Sulfate

Pyrimidine oligonucleotides can, when equipped with the thymidine-EDTA• Fe(II) analogue (T*), recognize and subsequently cleave double helical DNA at binding sites>15 base pairs in size. If binding affinities of unmodified oligonucleotides are to be determined under conditions relevant to those in vivo, alternate methods of detecting oligonucleotide-directed triple helix formation are required. The footprinting of short (up to 15 base pairs) triple helical regions on restriction fragment size DNA has been undertaken. Techniques for the determination of oligonucleo tide binding to double helical DNA using MPE•Fe(II), DNase I, and dimethyl sulfate have been developed. MPE•Fe(II) allows for the determination of binding site size, and has shown that oligonucleo tide binding to DNA is cation concentration, solvent, and oligonucleotide length dependent. DNase I footprinting was conducted under conditions optimal for DNase I activity (10 mM in each Mg^(+2) and Ca^(+2)), demonstrating that oligonucleotide-directed triplexes are capable of interfering with protein activity at the oligonucleotide binding site under physiological conditions, and that divalent cations can stabilize triple helix formation. Footprinting using dimethyl sulfate reveals that a single guanine 3' to the binding site becomes hyperreactive to methylation upon triplex formation. This suggests that the triplex-duplex junction involves a change in DNA conformation which is largely limited to a single base pair.

DMS footprinting reveals that the oligonucleotide CT-15 (T_5(CT)_5) does not bind the terminal 2 base pairs of the binding site in plasmid PDMAG10. DMS footprinting can be used to analyze the binding of oligonucleotides to DNA under conditions not amenable to MPE•Fe(II) or DNase I activity, and to assay the kinetics of oligonucleotide binding. DMS and DNase I footprinting techniques were used to assay for the effect of oligonucleotide concentration and base composition on binding affinity.

Chapter III: Oligonucleotide-Directed Triple Helix Formation using Oligonucleotides with Increased Binding Affinities

The specificity offered by the triple helix motif might provide a method for the artificial repression of gene expression and viral diseases. Changes in oligonucleotide structure could be used to control oligonucleotide affinity under in vivo conditions, where temporal and spatial intracellular pH (7.0-7.4) and ionic strength are strictly regulated and cannot be altered. Substitution at position 5 of pyrimidines alters the hydrophobic driving force, base stacking, and the electronic complementarity of the Hoogsteen base pairing for triple helix formation. Incorporation of 5-substituted pyrimidines offers a method of modulating binding affinity without changing the hydrogen bonding pattern and sequence specificities of pyrimidine oligonucleotides. Replacement of 2'd eoxycytidine with 5-methyl-2'-deoxycytidine increases the oligonucleotide affinity and extends the pH range for binding. Substitution of 5-bromo-2'deoxyuridine for thymidine increases binding affinity. Oligonucleotides constructed with 2'-deoxyuridine show lower binding affinities. Pyrimidine oligonucleotides constructed from 5-iodo-2'-deoxyuridines and 5-ethynyl-2'deoxyuridines display increased binding affinities relative to thymidine, but decreased relative to 5-bromouridine containing Oligonucleotides. Substitution by ethyl, pentyl, pentynyl, 2-phenyl-ethynyl, or fluoro at the 5 position of 2'deoxyuridine or bromo at the 5 position of 2'-deoxycytidine residues results in oligonucleotides with decreased binding affinities for double helical DNA.

Chapter IV: Efficient, Base-Specific Alkylation of DNA using N-Bromoacetyloligonucleotides

The attachment of a non-specific diffusible cleaving functionality to a DNA binding molecule allows for the elucidation of the structural principles for DNA recognition, a technique termed affinity cleaving. Once these principles have been determined, it becomes possible to design and attach structural domains designed to carry out a desired DNA modification. The development of a thymidine derivative capable of efficient and base specific DNA modification is reported. N-bromoacetyloligonucleotides are capable of near quantitative double strand modification of double helical DNA at a single guanine position in a manner which produces ends which are ligatable with compatible ends produced by conventional restriction enzyme digestion. The products thus produced are capable of transforming bacterial cell lines. N-bromoacetyloligonucleotides modify double helical DNA with specificities great enough to produce efficient (>90%) chemical cleavage at a single site within a yeast chromosome 340 kbp in size.

The acceleration obtained by tethering a reactive moiety to a DNA binding unit has been estimated. The rate of alkylation of DNA by N-bromoacetyloligonucleotides and bromoacetamide has been measured. Comparison of these ra tes indicates that an effective molarity of 2-3 M is obtained upon tethering the bromoacetyl moiety to an oligonucleotide to effect triple helix mediated DNA alkylation.

The utility of the bromoacetyl moiety as a reporter group is shown in studies concerning the effect of oligonucleotide length on binding affinity and the cooperative interaction between oligonucleotides binding abutting sites is reported.

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