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. My thesis work primarily focused 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 emerged.

At the beginning of the COVID-19 pandemic, shortages of the reagents for qPCR-based diagnostics led to the rationing of available tests. During this period, the Milam Lab proposed a flow cytometry-based approach for viral nucleic acid testing, which did not require the same reagents as qPCR-based methods. Viruses, like living organisms, possess characteristic nucleic acid sequences necessary for reproduction. The detection of these nucleic acid sequences can be used to test for the presence and quantification of their originating virus in a variety of applications. This research led to the development of a double stranded probe system which could be used for the sensitive and specific detection of viral nucleic acid sequences.

Double stranded probes (dsprobes) consist of a duplex with one fluorescently-modified strand (the probe sequence complementary to the target) and one quencher-capped strand. While the duplex is intact, the quencher prevents fluorescence; however, the target sequence displaces the quencher-capped strand, resulting in fluorescence signal. The probe/quencher system must be designed in such a way that hybridization within the initial duplex is stable, but still allows for displacement by the target sequence. This can be achieved with a single-stranded toehold region in the original dsprobe: the target sequence nucleates duplex formation in the single-stranded region, then completes duplex formation by displacing the quencher-capped strand.

Molecular beacons and dsprobes are often soluble species, therefore fluorescence intensity is measured for the bulk solution. Particle-immobilized probes, however, allow for more versatile experimental design. Commercial polystyrene microspheres offer covalent and noncovalent coupling routes for immobilizing probe sequences, facile centrifugation steps for target capture and separation, and multiple characterization tools to assess hybridization activity.