Synthesis, sequencing, and surveillance applications of xenonucleic acids

Abstract

The four nucleotides that form DNA (A, T, G, C) are central to the storage and propagation of genetic information. While nature has limited itself to four letters, these are not the only nucleotides capable of supporting the genetic code. Unnatural base pairing xenonucleic acids (ubp XNAs) are chemically synthesized nucleic acid analogs that base pair orthogonally to those found in nature. Ubp XNAs expand the accessible biochemical space of DNA, enabling innovation in diagnostics, therapeutics, and catalysts. Despite these advancements, the tools available for ubp XNA synthesis and sequencing remain decades behind those available for standard DNA. In this thesis, we develop tools for the synthesis and sequencing of ubp XNAs. We successfully demonstrate an enzymatic method for inserting a singular ubp XNA into a standard DNA context, and use this method to enable the sequencing of DNA with up to 12 letters. Next, we show that even without these tools, ubp XNAs can be used to overcome existing barriers in standard DNA biotechnologies. We develop an assay that uses two sets of non-standard bases to decrease off-target reactions in a multiplexed polymerase chain reaction (PCR). We benchmark the ability of this assay to detect 20 targets in three different matrices. Finally, we develop a proof-of-concept nucleic acid-based lateral flow platform for pathogen detection in low-resource settings that will integrate ubp XNAs to prevent non-specific hybridization. Together, these studies advance tools for ubp XNAs and demonstrate their abilities to support a new generation of biotechnologies.