Light-Activated Control of Translation by Enzymatic Covalent mRNA Labeling.

Activation of cellular protein expression upon visible-light photocleavage of small-molecule caging groups covalently attached to the 5' untranslated region (5' UTR) of an mRNA was achieved. These photocleavable caging groups are conjugated to in vitro transcribed mRNA (IVT-mRNA) through RNA transglycosylation, an enzymatic process in which a bacterial tRNA guanine transglycosylase (TGT) exchanges a guanine nucleobase in a specific 17-nucleotide motif (Tag) for synthetic pre-queuosine (preQ ) derivatives. The caging groups severely reduce mRNA translation efficiency when strategically placed in the 5' UTR.

Fluorescent turn-on probes for wash-free mRNA imaging covalent site-specific enzymatic labeling.

Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method currently suffers from a high nonspecific fluorescent background and is currently unsuitable for imaging RNA within complex cellular environments.

Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies.

Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation.

We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1.

Rapid access to phospholipid analogs using thiol-yne chemistry.

Phospholipids and glycolipids constitute an essential part of biological membranes, and are of tremendous fundamental and practical interest. Unfortunately, the preparation of functional phospholipids, or synthetic analogs, is often synthetically challenging. Here we utilize thiol-yne click chemistry methodology to gain access to phospho- and glycolipid analogs.