Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Bruce Eaton

Second Advisor

Robert Sievers

Third Advisor

Veronica Bierbaum

Fourth Advisor

Xiang Wang

Fifth Advisor

Ravinder Singh

Abstract

Despite the drastic decline in tuberculosis-related mortality due to the development of a vaccine and the discovery of effective anti-mycobacterial antibiotics during the middle part of the twentieth century, there has been a resurgence of the disease since the 1980's primarily caused by the emergence of HIV/AIDS and drug-resistant Mycobacterium tuberculosis strains. The need for improved diagnostic tests that are capable of greater specificity, sensitivity, and rapidity than the current tests is apparent and such tests would contribute significantly to the number of lives saved annually. A DNA aptamer-based biosensor has the potential to meet these challenges; aptamers have proven to have lower dissociation constants and longer binding times than many antibodies, and therefore may be capable of increasing the specificity and sensitivity of a biosensor. In addition, DNA may be modified with various functional groups capable of forming covalent bonds to target molecules, thus creating a new classification of target-binding oligonucleotides termed "reactamers." Such an addition would significantly reduce dissociation of the reactamer from the target. The systematic evolution of ligands by exponential enrichment (SELEX) process is commonly used to screen large pools of random oligonucleotides for the few sequences that can fold into a structure that is capable of binding a particular molecule. Aptamers and reactamers discovered in this way may be used to selectively remove the mycobacterium or antigen from a biological matrix, as well as serve as the detection molecule via conjugation with a reporter in a similar fashion as that of a sandwich-ELISA test. Using this process, aptamers and reactamers for a mycobacterial extracellular surface antigen, lipoarabinomannan (LAM), were isolated. These aptamers and reactamers represent initial, promising steps toward future development of oligonucleotides that can bind carbohydrates, which are notoriously difficult targets, with high affinity and specificity. The research therefore serves not only as an attempt to improve the tuberculosis diagnostic assay status quo, but also as an exploration of the multi-faceted nature of oligonucleotides and their various recognition, binding, and catalytic capabilities.

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