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Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

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Evaluating DFHBI-Responsive RNA Light-Up Aptamers as Fluorescent Reporters for Gene Expression.

Alicia Climent-Catala^1,2,3, Ivan Casas-Rodrigo⁴, Suhasini Iyer^1,5

¹Imperial College Centre for Synthetic Biology, London SW7 2AZ, U.K.

ACS Synthetic Biology

|November 22, 2023

Summary

Researchers explored RNA light-up aptamers as transcriptional reporters in E. coli, finding they offer faster dynamics than protein reporters but show higher population variability. These RNA reporters advance gene expression studies.

Keywords:

E. coli RNA light-up aptamers dynamics gene expression reporters synthetic biology

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Published on: October 19, 2017

Area of Science:

Molecular Biology
RNA Nanotechnology
Synthetic Biology

Background:

Protein-based fluorescent reporters are common but have limitations for dynamic or transcription-focused studies.
RNA nanotechnology offers functional RNA molecules as an alternative for transcriptional fluorescent reporters.
RNA aptamers can activate fluorescence upon binding small molecules, but their use as gene expression reporters needs characterization.

Purpose of the Study:

To investigate the performance of three RNA light-up aptamers (F30-2xdBroccoli, tRNA-Spinach, Tornado Broccoli) as transcriptional reporters in *Escherichia coli*.
To compare their suitability against a protein reporter in terms of activation range, cell growth effects, population variability, and dynamical behavior.
To assess their potential for advancing transcription-based studies and RNA-based circuits in bacterial systems.

Main Methods:

Time-course experiments were conducted to evaluate the activation range and impact on cell growth of RNA light-up aptamers.
Flow cytometry was used to compare single-cell level variability between RNA and protein-based reporters.
Dynamical behavior of RNA aptamers and protein reporters was analyzed and compared.

Main Results:

RNA light-up aptamers were confirmed as suitable transcriptional reporters over time in *E. coli*.
Expression of RNA light-up aptamers resulted in higher population variability at the single-cell level compared to protein reporters.
RNA light-up aptamers demonstrated potentially faster dynamics than fluorescent proteins in *E. coli*.

Conclusions:

RNA light-up aptamers represent a viable alternative to protein reporters for transcriptional studies in bacteria.
Their faster dynamics may provide advantages for studying biological processes with rapid changes.
Further implementation of these RNA reporters can enhance understanding of transcriptional processes and expand bacterial RNA-based circuits.