Mary Catherine Adams
Advisor: Prof. Valeria Milam

will defend a doctoral thesis entitled,

Competitive Binding of Synthetic Oligonucleotides

On

Friday, April 18 at 11:00 a.m.
MoSE Room 1224

Committee
            Prof. Valeria Milam – School of Materials Science and Engineering (advisor)
            Prof. Blair Brettmann– School of Materials Science and Engineering
            Prof. Seung Soon Jang – School of Materials Science and Engineering

    Prof. Rajesh Naik – School of Materials Science and Engineering, Adjunct appointment 
            Prof. M.G. Finn –  School of Chemistry and Biochemistry

Abstract

Nucleic acids evolved to serve a variety of biological functions, including the storage and transmission of the genetic code, the catalysis of protein synthesis, and the modulation of gene expression. Leveraging these properties, scientists are able to produce synthetic nucleic acid sequences capable of serving in a variety of non-genomic applications. This work primarily focuses on aptamers: single-stranded oligonucleotides capable of binding to non-nucleotide targets with high specificity and sensitivity. Aptamers can be used in a range of applications, including therapeutics, molecular sensing, and diagnostics. An aptamer’s target-binding behavior is a function of both its primary and secondary structure. The primary structure (identity and order of the bases), temperature, and solution conditions all determine the secondary structure (self-folded shape) of the oligonucleotide. 

The Milam Lab has developed a method for identifying aptamer sequences called Competition-Enhanced Ligand Selection (CompELS) which relies on competition between aptamer candidates. In order to better understand the CompELS process, I have developed a series of “barcoded” DNA sequences to screen for aptamers against the Fc fragment of the human IgG antibody; these barcodes can be used to monitor the changes in bound sequences over CompELS cycles. Following CompELS, both the “winning” (bound) sequences and the “losing” (unbound) sequences were analyzed, in order to identify any patterns that emerge.

Separate studies investigated the role of competitive binding of unlabeled SARS-CoV-2 RNA segments with double-stranded probes on microspheres initially in a signal-off state. Using high throughput analysis called flow cytometry, candidate probe systems were examined to find the best combination of stability (in the absence of target) and target responsiveness resulting in a transition to a signal-on state. Successful probes were identified along with unexpected long-range quenching effects from residual, closely-spaced probes remaining in the signal-off state. 

Collectively, this research helped us gain insight into the role of competition in both the aptamer selection process (CompELS) and oligonucleotide exchange events on microspheres.