DNA split aptamers as a biosensing platform for the detection of small drug molecules

honors thesisCollege of ScienceChemistryJennifer HeemstraPrescription drug overdose and abuse is a leading cause of death in the United States. It is a serious issue and has become increasingly problematic as opioids are being prescribed with a higher frequency. For this reason, fast, accurate detection of small drug molecules is crucial. The current standard for use in clinical drug detection is an enzyme‐linked immunosorbent assay (ELISA) that uses a series of antibodies to bind to the target drug and enable quantification via a colorimetric output. However, the antibodies used in an ELISA often cannot distinguish between similar molecules. They are generated in vivo, causing them to have a limited potential target scope as well as being costly. Deoxyribonucleic acids (DNA) can have a wide variety of functions outside of simply encoding genetic information. Aptamers are short sequences of DNA that are capable of binding target small‐molecules. They have emerged as a promising alternative to antibodies, as they are generated in vitro, where negative selections can be used to increase target selectivity. These aptamers can be cleaved to make split aptamers that only assemble in the presence of the target small molecule. In Chapter 1, we report a method of detection analogous to that of an ELISA. The cocaine split aptamer is used in conjunction with Split Aptamer Ligation (StAPL) technology. We attach one split aptamer fragment to a microplate, and in the presence of the target small molecule and the other aptamer fragment, the two aptamer fragments assemble. The reactive groups located on their ends undergo a cycloaddition reaction, covalently attaching the full sequence to the microplate. An attached biotin/streptavidin‐horseradish peroxidase complex allows for a colorimetric output upon addition of TMB substrate. We successfully used this system to detect varying concentrations of cocaine in buffer and biological fluids. In Chapter 2, we investigate the problem of limited numbers of split aptamers for small‐molecule targets. While there are many known aptamers, there are very few known split aptamers that bind small‐molecules. This research generates four new steroid binding split aptamers from their three‐way junction counterparts. We explore optimization, sequence changes, and selectivity of these new split aptamers. We successfully demonstrate a reliable method of separating aptamers with a privileged structure to generate new split aptamers for more targets. Finally, in Chapter 3, we report an effort to make our split aptamer ligation system compatible with qPCR. This would allow for a semi automatable system of detection, which would be more useful for potential clinical applications. In order to accomplish this, we alter the covalent linkage from a cycloaddition adduct to a morpholino linkage. This, as well as a primer binding region on the 5' end of one of the split aptamer fragments, allows the ligated split aptamer to be PCR readable. This is exciting progress in the effort toward creating a semi automatable system of detection for small drug molecules using split aptamers