Date Approved

2019

Degree Type

Campus Only Thesis

Degree Name

Master of Science (MS)

Department or School

Chemistry

Committee Member

Jeffrey Guthrie, Ph.D.

Committee Member

Hedeel Evans, Ph.D.

Committee Member

Heather Holmes, Ph.D.

Abstract

Aptamers are short, synthetic oligonucleotide sequences that can selectively bind to targets with high affinity. Selection of aptamers is accomplished by the Systematic Evolution of Ligands by EXponential enrichment (SELEX). The target is incubated with a random sequence oligonucleotide library, and target-bound sequences are separated from the unbound pool to generate a higher affinity library for use in subsequent selection rounds. Capillary electrophoresis (CE) has been used for the selection of large protein targets and requires fewer rounds of selection than conventional SELEX methods. Unlike large targets, small molecule targets bound to DNA sequences show little to no change in electrophoretic mobility, resulting in poor separation from the unbound sequences. To solve this problem, quantum dots (Qdots) with covalently bound target molecules can be employed to effectively increase the size of small molecular targets and allow efficient separation based on the Qdots’ mobility. As proof of concept for the use of Qdots in CE-SELEX, we used commercially available Qdots conjugated with streptavidin to show that Qdot-target-DNA complexes can be separated from unbound DNA sequences. Laser-induced fluorescence-CE and fluorescently labeled DNA allowed for real-time identification of DNA-target binding by fluorescence resonance energy transfer between the DNA fluorophore and the Qdots, changing the latter's fluorescent intensity. After five rounds of selection, including a negative selection round to avoid quantum dot-binding sequences, the final aptamer library was determined to have a dissociation constant of 1.9 nM for the streptavidin target.

Download

Share

COinS