UC San Diego

The importance of nucleic acids has expended past their role in encoding life and into a space of scientific utility and discovery. Developing new, robust methods to study and manipulate them in their natural environments and harness their programmability for fundamental research tools and biomedical applications has become a critical goal for researchers. This work presents an enzymatic tool for functional modification of single stranded DNA oligonucleotides as well a strategy to assemble functional nucleic acid protein conjugates.

Due to its greater stability and ease of synthesis, DNA, rather than RNA, is the nucleic acid of choice for many functional applications. Often, these applications require the DNA oligonucleotide to be modified in some way with an affinity tag or reporter. There are a limited number of methods to label DNA oligonucleotides, all of which have their shortcomings, including being cost prohibitive, inefficient, or a limited small molecule scope. In this dissertation I develop an enzymatic single stranded DNA modification method, expanding on the powerful RNA transglycosylation at guanine (RNA-TAG) modification tool developed in our lab. DNA-TAG offers researchers with an unprecedented tool to generate modified oligonucleotides in their lab. This work describes the rational and iterative design of DNA-TAG and demonstrates its utility through fluorescent northern blot and RNA fluorescent in situ hybridization (FISH).

Combining the programmability of nucleic acids with the functions of proteins has the potential to enable powerful assemblies with unique and critical functions, both natural and synthetic. Developing novel strategies to bring these classes of biomolecules together in a robust and specific manner continues to facilitate new applications. Here I present a fully enzymatic method to covalently assemble a functional RNA-Protein conjugate using a novel bifunctional small molecule probe and demonstrate he utility of the system by recruitment of an endonuclease to an RNA of interest to induce degradation.

With the herein presented advancements, we expand the application space of nucleic acid modification. Nucleic acid transglycosylation at guanine (NA-TAG) technologies provide researchers with a single tool capable of modifying nearly any single stranded nucleic acid substrate with a small molecule of their choice for downstream applications.