Chemo-Enzymatic Strategies to Functionalize RNA with Biophysical Probes Using Template Dependent and Independent Polymerases

Abstract

In the hall of fame of biopolymers, RNA forms diverse structures which enable it to perform an extensive array of functions ranging from data storage to catalysis. The broad range of RNA structures in cellular milieu enables it to bind to various metabolites, proteins and nucleic acids. Unraveling these structures and its associated functions are crucial for understanding various disease states and developing diagnostic tools. Several biophysical tools like fluorescence, NMR, EPR and X-ray crystallography are conveniently used to study the structure of RNA. However, RNA has no intrinsic biophysical read-out, which makes it imperative to tag RNA with extrinsic functional probes. Traditionally used chemical methods can be employed to functionalize RNA but is limited by several drawbacks. The thesis describes the development of various innovative chemo-enzymatic strategies for labeling RNA with biophysical probes using template dependent and independent polymerases. In the initial part of the thesis, we demonstrate the incorporation of a vinyl tag into RNA oligonucleotides (ONs) using in vitro transcription reaction. Further, we show the propensity of functionalizing this vinyl moiety on RNA by oxidative Heck reaction, employed in constructing fluorogenic RNA ONs labeled with various heterocyclic biophysical probes. Also, the vinyl moiety is compatible for a reagent-free inverse electron-demand Diels-Alder reaction. Given that transcription mediated incorporation of functional tags has its own drawbacks due to indiscriminate RNA labeling, we devised a novel site-specific chemo-enzymatic strategy to label RNA at its 3′ end utilizing the promiscuity of terminal uridylating enzyme, SpCID1. Employing the enzyme, we introduced a range of bioorthogonal click-compatible tags on RNA, which is fine-tuned to incorporate single or multiple functional moieties. Notably, we developed a novel technology namely, sgRNA-Click (sgR-CLK), wherein we repurposed the uridylating enzyme to introduce multiple clickable handles on CRISPR sgRNA. Employing the minimally perturbing azide tags on sgRNA as a ‘trojan horse’ on CRISPR-dCas9 system, we demonstrated site-directed display of small molecules on target genes. Further, we also present various applications of this labeling technology, which can be used to introduce FRET pairs and site-specific internal click labels on RNA. Finally, we establish the incorporation of microenvironment responsive nucleoside analogues enzymatically into RNA using SpCID1, which helps in deconvoluting real-time substrate binding to this terminal uridylating enzyme. Overall in the thesis, we provide a brief introduction on strategies to label RNA, followed by an in depth discussion on chemo-enzymatic approaches we developed to functionalize RNA with biophysical probes, and lastly, the application of these novel labeling technologies.