New approaches for sensing metabolites and proteins in live cells using RNA

Abstract

Tools to study the abundance, distribution, and flux of intracellular molecules are crucial for understanding cellular signaling and physiology. Although powerful, the current FRET-based technology for imaging cellular metabolites is not easily generalizable. Thus, new platforms for generating genetically encoded sensors are needed. We recently developed a new class of biosensors on the basis of Spinach, an RNA mimic of GFP. In this case, RNA aptamers against a target ligand are modularly fused to Spinach that substantially induce Spinach fluorescence in the presence of ligand. We have used this approach to detect metabolites and proteins both in vitro and in living bacteria, thus providing an alternative to FRET-based sensors and a generalizable approach for generating fluorescent sensors to any ligand of interest.

Figure 1. Modular strategy for Spinach-based sensors.<

br>(a) Spinach is an aptamer that binds a small molecule dye called DFHBI (green ball). Both DFHBI and Spinach are nonfluorescent until binding occurs, which turns on fluorescence of the Spinach-DFHBI complex. Stem loop 2 of Spinach was found to tolerate insertion of additional sequences, and is the region that is modified to generate sensors. (b) To generate sensors, Spinach is modified to include a transducer region (orange) and a recognition module (blue). The recognition molecule is typically an aptamers generated against a target ligand by SELEX. Transducer length and composition can be varied for optimal sensor function. (c) In the absence of DFHBI and ligand (purple hexagon), the Spinach-based sensor displays minimal fluorescence. However, upon target binding, the recognition module of Spinach folds, which induces overall folding. The Spinach-based sensor can then bind DFHBI and activate fluorescence. This general strategy can be used to generate sensors against a vast array of targets.

Figure 2. A Spinach-based sensor of SAM

(a) A SAM sensor shows minimal fluorescence in the absence of SAM, even in the presence of DFHBI. However, upon SAM addition, fluorescence is activated over 20-fold. (b) The SAM sensor is specifically activated by SAM, and not the closely related molecules S-adenyl histidine (SAH), methionine, or vehicle. (c) Fluorescence activation occurs rapidly for the SAM sensor and reaches saturation on the order of minutes. (d) Fluorescence activation is reversible, and diminishes on the order of minutes for the SAM sensor after SAM has been removed. These data are representative of the metabolite and protein sensors generated based on Spinach, and are reproduced with permission from ref. .

Figure 3. SAM levels can be imaged in living cells.<

br>(a) E. coli expressing the SAM sensor were imaged after treatment with methionine. Time course imaging revealed distinct patterns of SAM accumulation among individual cells. Some cells exhibited slow SAM accumulation (arrowhead) while others had higher than average (arrow) increases. A double arrow highlights a cell that initially increases then decreases SAM level. Pseudocolored images show the fold increase in fluorescence at each time point relative to 0 min (0- to 11.2-fold). Scale bar, 5 um. (b) Quantification of SAM levels in 800 individual cells revealed the large variance in SAM abundance after treatment with methionine, highlighting the importance of being able to monitor processes on the single-cell level. These data are reproduced with permission from ref. .