Date of Graduation

Summer 2010

Degree

Master of Science in Chemistry

Department

Chemistry

Committee Chair

Bhaskar Datta

Keywords

selex, aptamers, oligonucleotide, chromophores, fluorophores, nucleic acids

Subject Categories

Chemistry

Abstract

The Systematic Evolution of Ligands by EXponential enrichment (SELEX) process has allowed the discovery of aptamers with the ability to bind target molecules with high affinity and specificity. This opens up avenues to develop easy, selective and cost-effective methods to monitor aptamer-target interactions. Conventional optical assays involve the functionalization of the termini of an oligonucleotide with a fluorophore and a quencher for detecting analytes. In this work, i utilized nucleic acid staining reagents as chromophores to develop a signaling strategy that avoids synthetic modification of aptamers. Three different aptamers (thrombin-, theophylline-, and ATP-binding aptamers) that span a range of sizes, secondary structures and affinities were employed for this work. I monitored the formation of the fluorescent complex by nucleic acid and chromophore in the presence and absence of a cognate target. My study reveals that the chromophores SYBR Green I and thiazole orange (TO) can be used across RNA and DNA aptamers, albeit with different efficiencies in each specific case. The efficiency of my strategy depends on the binding sites on the aptamer accessible to the chromophore, the binding affinity of the chromophore for the aptamer, and the affinity of the aptamer for its cognate target.

Copyright

© Kwabena Amofa Nketia Sarpong

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