Fluorescent turn-on probes are important tools for the investigation of biochemical processes and diagnosis of disease markers because they are sensitive and easy to observe. Among many biomacromolecules, the detection of RNAs is one of the greatest challenges because of the dynamic and short-lived nature of the RNAs and the lack of chemical probes and methods that can be used to selectively distinguish them. In our previous communication, we demonstrated the site-specific incorporation of PreQ1, a natural substrate of bacterial tRNA guanine transglycosylase (TGT), bearing fluorophores into a short RNA motif via a transglycosylation at guanosine (RNA-TAG). By using thiazole orange (TO) modified PreQ1, we observed a 40-fold increase of fluorescent signal with the RNA hairpin and 3-fold increase when the RNA hairpin motif is incorporated into the 3’ UTR of an mRNA transcript. In this study, we aim to chemically modify the existing thiazole orange fluorogenic probes to improve the fluorescent turn-on and achieve improved reaction kinetics. We synthesized an array of PreQ1-TO derivatives that can be accepted by the TGT. Using a newly synthesized PreQ1-TO, we observed 100-fold and 10-fold of fluorescent enhancements upon the enzymatic transglycosylation reaction with the RNA hairpin and the hairpin modified mRNA transcript, respectively. We also observed improved kinetics by substituting and existing polyethylene glycol linker with an alkyl linker. We believe these improvements will better enable more sensitive applications such as live cell imaging and the detection of early stage diseases.

Synthesis and structure-activity relationship study of PreQ1-thiazole orange fluorogenic probes for RNA-TAG

251st National Meeting (2016)

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