Genetically Encoded Ratiometric RNA‐Based Sensors for Quantitative Imaging of Small Molecules in Living Cells

Abstract

Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA‐based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB‐to‐Broccoli fluorescence ratio to quantify the cell‐to‐cell variations of target concentrations. These ratiometric sensors can be broadly applied for live‐cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.

Live‐Cell Imaging of Guanosine Tetra‐ and Pentaphosphate (p)ppGpp with RNA‐based Fluorescent Sensors**

“…To better normalize the cell‐to‐cell variation in the RNA sensor expression level, we cloned S2 in a pET28c vector together with a DNB RNA aptamer tag (Table S1). Here, the DNB/TMR‐DN signal can be used as a reference to calibrate the cellular expression levels of Broccoli‐based sensors [36] . To test this new ratiometric RNA sensor, we transformed the S2‐DNB conjugate into one batch of BL21 cells and S2‐DNB with RelA* gene into another.…”

Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules

“…To solve this problem, we have recently developed a type of ratiometric RNA-based sensors using an orthogonal pair of RNA/fluorophore conjugates, Broccoli/DFHBI-1T (λ ex /λ em , ∼480/503 nm) and DNB/SR-DN (λ ex /λ em , ∼571/591 nm). 25 The DNB aptamer can specifically bind with a quencher moiety, dinitroaniline (DN). DN is an efficient contact quencher for a variety of S2).…”

Live‐Cell Imaging of Guanosine Tetra‐ and Pentaphosphate (p)ppGpp with RNA‐based Fluorescent Sensors**

“…Here, the DNB/TMR-DN signal can be used as a reference to calibrate the cellular expression levels of Broccoli-based sensors. [36] To test this new ratiometric RNA sensor, we transformed the S2-DNB conjugate into one batch of BL21 cells and S2-DNB with RelA* gene into another. Indeed, clear (p)ppGppinduced S2 signal was observed in the RelA* strain, and the DNB fluorescence was quite similar in both strains (Figure 5 a).…”