Chemoenzymatic labeling of RNA using terminal uridylyl transferase and bioorthogonal click chemistry.
The ability of native and engineered nucleic acid-processing enzymes to incorporate clickable nucleotide substrates has greatly advanced bioorthogonal labeling of nucleic acids, overcoming the limitations of conventional solid-phase oligonucleotide (ON) synthesis. In this chemoenzymatic approach, template-dependent polymerases routinely enable the incorporation of nucleotides bearing small reactive handles. The resulting nucleic acids undergo chemoselective reactions, such as azide-alkyne cycloaddition, inverse-electron-demand Diels-Alder, or Staudinger ligation, to install desired functionalities. Alternatively, the promiscuity of template-independent transferases, such as terminal uridylyl transferase (TUTase), provides access to site-specific labeling of RNA ONs at the 3'-end. In this methods chapter, we detail protocols for incorporating azide-modified UTP analogs into short RNA ONs and highly structured CRISPR guide RNAs (sgRNAs) using the terminal uridylyl transferase SpCID1. We describe methods to control the enzyme's incessant incorporation behavior and enable subsequent click functionalization of the RNAs. Finally, we demonstrate remodeling of the CRISPR system via synthesis of azide-modified sgRNAs, which when complexed with dCas9, recruit azide groups to specific gene targets for post-hybridization functionalization.
Dutta S, Pal A, Srivatsan SG