The Application of Oligonucleotide Templated Chemical Reactions to DNA Aptamers

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Creator: 

Mattice, Christopher Matthew Colin

Date: 

2018

Abstract: 

The ability of an aptamer to catalyze a chemical reaction under selective conditions presents a novel avenue for the exploration of biosensors and molecular payload delivery. To date, limited research exists for pairing oligonucleotide-templated reactions with the selective nature of DNA aptamers. A system can be designed wherein the conformational change in aptamer structure associated with target binding brings two previously spatially isolated reactants into proximity, thereby catalyzing their reaction through an increase in effective molarity. A rationally designed aptamer-mediated SN2 displacement of a sulfonyl-based fluorescence-quencher resulted in an effective increase in fluorescence upon mixing of the aptamer with two appropriately modified complementary oligonucleotides. This increased fluorescence could be slowed by the presence of the aptamer target, permitting the development of an aptamer-based sensor for ochratoxin A with a linear dynamic range of 100 μM to 100 pM and a limit of detection of 1.5 pM.

Similar aptamer-based sensor systems were also developed that could take advantage of the fluorogenic copper catalyzed azide-alkyne cycloadditions between two labelled probes controlled by the aptamer target. This has been demonstrated using the thrombin structure switching aptamer to produce a linear response to thrombin between 100 nM and 10 pM with a limit of detection of 3.9 fM. These fluorogenic click modifiers were also incorporated directly into a structure switching thrombin aptamer such that the G-quadruplex formed upon thrombin binding brings the two modifiers into proximity. This constitutes a turn-on sensor for thrombin, with detection of thrombin between 1 μM and 1 nM possible.

As a final proof of concept, the fluorogenic azide and alkyne modifiers were incorporated on either side of a split aptamer for cocaine, such that the presence of cocaine will result in a detectable fluorescent signal from ligation of the split aptamer sequences. Using this system, a qualitative cocaine sensor in the range of 100 μM to 10 pM was established. This project has demonstrated the first steps towards controlling chemical reactions using DNA aptamers. Having demonstrated preliminary functionality through these applications, future applications in drug delivery, enhanced sensors, and selective chemical synthesis constitute intriguing new avenues in aptamer research.

Subject: 

Biochemistry
Organic Chemistry
Analytical Chemistry

Language: 

English

Publisher: 

Carleton University

Thesis Degree Name: 

Doctor of Philosophy: 
Ph.D.

Thesis Degree Level: 

Doctoral

Thesis Degree Discipline: 

Chemistry

Parent Collection: 

Theses and Dissertations

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