Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Ivan Dmochowski

Abstract

Photoactivatable oligonucleotides are DNA or RNA molecules whose function is temporarily blocked or "caged" by a chemical moiety that can be activated by light. By using light to turn on activity, high spatial and temporal control of oligonucleotide function can be attained. We have developed novel techniques to cage oligonucleotides using light-activatable groups that introduce a strand break upon photolysis. One method of inhibiting gene expression involves the use of catalytic DNA strands known as DNAzymes, specifically ones with the ability to cleave RNA. We have demonstrated efficient photomodulation of a biotinylated split DNAzyme by employing streptavidin to disrupt the active conformation. Light-controlled split DNAzymes could potentially be a tool for studying gene expression in vitro. Additionally, a novel photochemical approach for isolating mRNA from single neurons was developed by creating hairpin-bandage hybrids. This design utilizes a photoactivatable oligonucleotide hairpin that targets the poly(A)+ tails of mRNA. The antisense strand of the hairpin is linked via a photocleavable spacer to the blocking strand, which is divided into two parts by a second photocleavable spacer. Experiments in single neurons have confirmed the utility of hairpin-bandage hybrids for fluorescently monitoring oligonucleotide dissociation in vivo and for isolating of mRNA from single cells.

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