| Description |
The development of systematic evolution of ligands by exponential enrichment, or SELEX, in 1990 propelled interest in nucleic acid engineering. The ability of nucleic acids to undergo directed evolution has resulted in the widespread interest in the development of aptamers, or single stranded nucleic acids that possess affinity to a specific target molecule. Aptamers exist to diverse classes of molecules, and because of their versatility are being regarded as robust recognition elements for a variety of biotechnology applications. Further, the small-molecule harnessing potential of aptamers has shown great promise for the development of biosensors. Importantly, traditional selection methods can be expanded upon to enable the generation of functional nucleic acids which are optimized for biosensing platforms. Novel SELEX methods are a powerful tool to develop innovative technologies for detection and imaging using aptamer-based biosensors. Our lab has contributed to a variety of areas within this field including the use of ribozymes as a tag for intracellular imaging of RNA. We have designed a novel IP-SELEX method which allows for the generation of ribozymes that are capable of performing a reaction to covalently attach a small-molecule fluorophore to itself (Chapter 2). We show that this ribozyme can label in cellular conditions and hold great promise as a genetically encodable tag for live cell labeling of mRNA. We have also developed a new class of small-molecule sensors through the evolution of nucleic acids with noncanonical backbone structures, or xeno nucleic acids (XNA). XNA molecules have many advantageous properties because of their noncanonical structures, notably nuclease resistance. Using our unique method, optimized by the use of an XNA primer, we were able generate the first artificial genetic polymers capable of small-molecule recognition (Chapter 3). We discovered threose nucleic acid (TNA) aptamers with affinity for a small-molecule mycotoxin (OTA). We show that these aptamers have outstanding biostability in the presence of nucleases and retain the ability to bind the target in these environments. We acknowledge the potential for structure-switching (SS) aptamer biosensors as a privileged architecture for small-molecule detection and the development of fluorescence assays. We propose an innovative technique for the direct selection of SS biosensors, in which we take advantage of both restriction digests and polymerase chain reaction (PCR) as key steps to eliminate nonfunctional sequences. Importantly, because amplification simultaneously serves as the selection step there is no requirement for a solid support. We anticipate this will overcome the limitations of bead-based selection methods and enable for efficient and effective generation of SS biosensors. |
