RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP

Abstract

Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.

Figure 1

RNA-based fluorescent biosensors detect cyclic di-nucleotide second messengers. (a) Design scheme for a fluorescent biosensor that detects cyclic di-nucleotides. Ligand binding to the Vc2 riboswitch aptamer (blue) enables the Spinach aptamer (black) to bind and activate the conditionally fluorescent molecule DFHBI. (b) Sequence and secondary structure model of the Vc2-Spinach construct. Mutants analyzed in the study are boxed. C-di-GMP is shown in purple. Spinach stem loops are numbered in orange. (c) In vitro screen of Vc2-Spinach biosensor variants for fluorescence activation in response to cyclic di-nucleotides. Error bars represent the standard deviation of three independent experiments with duplicate samples.

Figure 2

WT Vc2-Spinach is a sensitive biosensor for c-di-GMP in live cells. (a) In vitro analysis of WT Vc2-Spinach binding affinity for c-di-GMP. Data from three independent replicates and the best-fit curve are shown. Background fluorescence (without c-di-GMP) was subtracted from all data points. (b) Differential interference contrast (DIC) and fluorescence images of E. coli cells expressing Vc2-Spinach tRNAs and WspR enzyme variants after incubation with DFHBI. Cells expressing WT WspR were observed to have more cellular debris compared to other samples. Scale bars represent 10 μm. (c) Quantitation of mean fluorescence intensity of cells. Error bars indicate S.E.M. for at least 50 cells. P-values from student's t-test comparisons for fluorescence changes discussed in text are shown.

Figure 3

Analysis of the enzymatic activity of DncV in live cells using Vc2-Spinach biosensors. (a) In vitro analysis of G20A Vc2-Spinach binding affinity for c-di-GMP (black) and c-AMP-GMP (red). Data from three independent replicates each and the best-fit curves are shown. Background fluorescence (without ligand) was subtracted from all data points. (b) Differential interference contrast (DIC) and fluorescence images of E. coli cells expressing Vc2-Spinach tRNAs and DncV enzyme variants after incubation with DFHBI. Scale bars represent 10 μm. (c) Quantitation of mean fluorescence intensity of cells. Error bars indicate S.E.M. for at least 50 cells. P-values from student's t-test comparisons are shown.