Publication Date

2015-07-30

Availability

Embargoed

Embargo Period

2016-07-29

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Chemistry (Arts and Sciences)

Date of Defense

2015-06-30

First Committee Member

Sapna K. Deo

Second Committee Member

Leonidas Bachas

Third Committee Member

Angel Kaifer

Fourth Committee Member

Pirouz Daftarian

Abstract

Nucleic acids can be pointed biomarkers for a variety of diseases, medical disorders, and even injuries. Their importance is ever growing in the modern clinical setting, which is seeing rapid development in technologies, such as high-throughput sequencing of genomic content, utilizing large data sets to make new correlations, ultimately leading to the discovery of more novel biomarker targets. In the wake of these rapidly accumulating discoveries, there is a need for the creation of biosensor technologies capable of distilling the data-heavy discoveries into clinically applicable diagnostic tests. Herein, work pertaining to the development of nucleic acid biosensors, and the continued improvement of the bioluminescent proteins upon which they are based, is discussed. In particular, bioluminescent stem-loop probes utilizing Renilla luciferase as a reporter are described for the detection of nucleic acid targets, and demonstrated in detecting a miR-21 microRNA target from human serum samples. To improve the sensitivity and utility of these bioluminescent probes, the genetic alteration of Gaussia luciferase to produce truncated variants exhibiting unique spectral and kinetic characteristics is discussed, as well as the bacterial expression of a truncated Vargula luciferase for high-throughput detection platforms. Finally, the prospective research pertaining to the overlap of both of these areas is highlighted, as new bioconjugation techniques are developed for the chemical synthesis of these bioluminescent sensing systems.

Keywords

nucleic acids; biosensors; bioluminescence; protein expression; bioconjugation

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