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.

Developing a Fluorescent Toolbox To Shed Light on the Mysteries of RNA.

Technologies that detect and image RNA have illuminated the complex roles played by RNA, redefining the traditional and superficial role first outlined by the central dogma of biology. Because there is such a wide diversity of RNA structure arising from an assortment of functions within biology, a toolbox of approaches have emerged for investigation of this important class of biomolecules. These methods are necessary to detect and elucidate the localization and dynamics of specific RNAs and in doing so unlock our understanding of how RNA dysregulation leads to disease.

A Bioorthogonal Near-Infrared Fluorogenic Probe for mRNA Detection.

There is significant interest in developing methods that visualize and detect RNA. Bioorthogonal template-driven tetrazine ligations could be a powerful route to visualizing nucleic acids in native cells, yet past work has been limited with respect to the diversity of fluorogens that can be activated via a tetrazine reaction. Herein we report a novel bioorthogonal tetrazine uncaging reaction that harnesses tetrazine reactivity to unmask vinyl ether caged fluorophores spanning the visible spectrum, including a near-infrared (NIR)-emitting cyanine dye.

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.

Electrochemical Control of Rapid Bioorthogonal Tetrazine Ligations for Selective Functionalization of Microelectrodes.

We demonstrate that bioorthogonal tetrazine ligations can be utilized to rapidly modify electrode surfaces, both with redox probes and enzymes. Furthermore, we show that the redox-active nature of 1,2,4,5-tetrazines can be exploited to gain electrochemical control over surface modification. To our knowledge this is the first demonstration of controlling a tetrazine ligation by changing the redox state of one of the reactants.

Interactions of AsCy3 with cysteine-rich peptides.

There is great interest in fluorogenic compounds that tag biomolecules within cells. Biarsenicals are fluorogenic compounds that become fluorescent upon binding four proximal Cys thiols, a tetracysteine (Cys(4)) motif. This work details interactions between the biarsenical AsCy3 and Cys(4) peptides.