The detection and manipulation of RNA is greatly aided by chemical modification. Therefore, there is tremendous interest in novel methods to site-specifically associate RNA with small molecules such as imaging probes and affinity labels. Conventional methodologies for detecting RNA include the use of antisense probes, aptamers, and fusion proteins that recognize specific RNA secondary structures. Relatively less explored are the use of enzymatic reactions for site-specific RNA labeling. Notably, these past approaches have not demonstrated the ability to append large functional molecules directly onto the RNA of interest. Instead they typically rely upon small bio-orthogonal handles, which after undergoing a second chemical reaction, can be modified by functional probes such as fluorophores or affinity ligands.
An ideal enzymatic reaction for labeling RNA would involve recognition of a minimal RNA structural motif, result in irreversible covalent modification, and would be capable of directly incorporating a diverse array of functional molecules such as fluorophores, affinity ligands, etc. in a single step.
Chemists from UC San Diego have addressed this need by developing an enzymatic method (RNA-TAG; transglycosylation at guanosine), to directly append large functional molecules site-specifically to RNA.
This method utilizes a bacterial enzyme (tRNA guanine transglycosylase; TGT) to exchange specific guanine nucleobases with functional derivatives of the bacterial nucleobase PreQ1. Any RNA with this particular hairpin structure, even relatively large transcripts of genes, can be tagged in this way, which should prove useful in a wide variety of biomedical and biochemical applications.
This patent-pending RNA-TAG methodology has applications for imaging, and may also play a key role in discovering new RNA-protein interactions, drug screening applications, and exploring RNA lifetime dynamics.
|United States Of America||Published Application||20180148764||05/31/2018||2015-196|
|Patent Cooperation Treaty||Published Application||2016141243||09/09/2016||2015-196|
Additional Patent Pending