The use of modified oligonucleotides to investigate biological applications for triple helix formation

Citation

Hacia, Joseph Gerard (1995) The use of modified oligonucleotides to investigate biological applications for triple helix formation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/15j7-1b14. https://resolver.caltech.edu/CaltechETD:etd-10122007-074500

Abstract

Oligonucleotide-directed triple helix formation was assayed for potential biological applications. Unmodified pyrimidine-rich oligodeoxyribo-nucleotides were found to block the progress of primer extension by DNA polymerase in vitro through triple helix formation. Klenow fragment polymerization was obstructed at sites that map near the proximal boundary between duplex and triplex. Among a family of related three-stranded structures, longer triplexes were more effective polymerase inhibitors than shorter complexes. One such complex provided an effective polymerase blockade for at least twenty minutes. Chemical modifications enhanced both triple helix formation and the nuclease resistance of pyrimidine-rich and purine-rich oligonucleotides. Unmodified purine-rich and pyrimidine-rich oligonucleotides containing a diastereomeric mixture of phosphorothioate or stereoregular (all R[rho]) phosphorothioate linkages were tested for triple helix formation by quantitative DNase I footprinting analysis. Both purine-rich and pyrimidine-rich phosphorothioate oligonucleotides containing modified nucleosides formed triple helical complexes in vitro under physiological ionic conditions. Pyrimidine-rich oligoribonucleotides as well as 2'-OMe oligoribonucleotides bound DNA with high affinity. Only purine-rich oligodeoxyribonucleotides had significant affinity for double-helical DNA. Small uridine-rich oligoribonucleotides were overexpressed in E. coli for in vivo triple helix formation studies using a low-copy number plasmid target site. Micromolar intracellular concentrations of intact oligoribonucleotide were generated; however, in vivo footprinting analysis indicated that no triple helical complexes formed. [Beta]-galactosidase activity assays further indicated that the presence of the uridine-rich RNA did not inhibit transcription of a chimeric lacZ gene in which the triple helix target site was inserted. Modified nuclease-resistant pyrimidine-rich and purine-rich oligonucleotides were tested for triple helix formation on Xenopus oocyte minichromosomes. In vivo footprinting analysis showed that none of the oligonucleotides formed triple helical complexes with the target site. A modified oligonucleotide, which directed an alkylation reaction to a specific guanosine residue on duplex DNA in vitro, did not react in vivo with the target minichromosomes. Further chemical modifications will have to be made to facilitate in vivo triple helix formation.

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