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.
