Abstract
RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.
Highlights
RNAs mediate many essential processes in live cells, both as the messenger RNA that transcribes genetic information to protein and as non-coding RNAs that regulate gene expression at the transcriptional and post-transcriptional level[1,2]
Malachite green aptamer (MGA) fluorescence is enhanced by aptamer scaffolds
The MGA scaffolds were integrated into a pET28a plasmid, which allowed MGA’s in-vitro and cellular expression to be regulated by the T7 promoter and lac operator system respectively
Summary
RNAs mediate many essential processes in live cells, both as the messenger RNA (mRNA) that transcribes genetic information to protein and as non-coding RNAs that regulate gene expression at the transcriptional and post-transcriptional level[1,2]. Malachite green aptamer (MGA) is one of the first fluorescence-activating aptamers to be described in the literature[12,13] It was identified through in-vitro selection for its high binding affinity and strong fluorescence intensity. We explore the expression of a scaffold consisting of 6 copies of MGA in bacterial cells, showing a compatible spectrum of fluorescence with other currently available RNA aptamers. We observe a strong and stable MGA fluorescence that can be linked to multiple kinds of RNA molecules without significant fluorescent signal loss in E. coli This MGA fluorescence can be studied using microscopy and flow cytometry showing its potential as a tool to study RNA expression and dynamics in-vivo
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
