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 extensivearray of functions ranging from data storage to catalysis The broad range of RNA structures in cellularmilieu enables it to bind to various metabolites proteins and nucleic acids Unraveling these structures andits 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 usedto study the structure of RNA However RNA has no intrinsic biophysical read out which makes itimperative to tag RNA with extrinsic functional probes Traditionally used chemical methods can beemployed to functionalize RNA but is limited by several drawbacks The thesis describes the developmentof various innovative chemo enzymatic strategies for labeling RNA with biophysical probes using templatedependent and independent polymerases In the initial part of the thesis we demonstrate the incorporationof a vinyl tag into RNA oligonucleotides ONs using in vitro transcription reaction Further we show thepropensity of functionalizing this vinyl moiety on RNA by oxidative Heck reaction employed inconstructing fluorogenic RNA ONs labeled with various heterocyclic biophysical probes Also the vinylmoiety is compatible for a reagent free inverse electron demand Diels Alder reaction Given thattranscription mediated incorporation of functional tags has its own drawbacks due to indiscriminate RNAlabeling we devised a novel site specific chemo enzymatic strategy to label RNA at its 3 8242 end utilizing thepromiscuity of terminal uridylating enzyme SpCID1 Employing the enzyme we introduced a range ofbioorthogonal click compatible tags on RNA which is fine tuned to incorporate single or multiplefunctional moieties Notably we developed a novel technology namely sgRNA Click sgR CLK whereinwe newline newline