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

Article abstract of DOI:10.1021/ja311960g

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.

Full text of DOI:10.1021/ja311960g

Communication
pubs.acs.org/JACS
RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second
Messengers Cyclic di-GMP and Cyclic AMP-GMP
Colleen A. Kellenberger,† Stephen C. Wilson,† Jade Sales-Lee,† and Ming C. Hammond*,†,‡
‡
†Department of Chemistry and Department of Molecular & Cell Biology, University of California, Berkeley, California 94720,
United States
S
* Supporting Information
to Spinach. Furthermore, mutation of the ligand binding pocket
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.
of the riboswitch enables recognition of both c-AMP-GMP and
c-di-GMP. Finally, we demonstrate the utility of RNA-based
biosensors for detecting the activity of cyclic dinucleotide
producing enzymes in live cells and validate the cellular activity
of the first discovered c-AMP-GMP synthase, DncV.
Binding of the conditionally fluorescent molecule DFHBI by
the Spinach aptamer is highly dependent on formation of its
second stem loop.12 We replaced this stem loop with the
aptamer domain of the Vc2 GEMM-I class riboswitch that
binds c-di-GMP with very high affinity (Kd ∼ 0.01 nM) and
specificity (Figure 1a,b).13,14 The natural P1 stem of Vc2 was
used directly as the transducer stem, since published structural
probing experiments were consistent with P1 stabilization upon
ligand binding.13 We found that this RNA construct, called
yclic dinucleotides are a newly expanded class of second
Cmessengers that mediate intracellular signaling pathways
in bacteria.1 Cyclic di-GMP (c-di-GMP) has been shown to
regulate physiological processes such as biofilm formation,
motility, and virulence response.2 More recently, two more
cyclic dinucleotides were identified as natural products. Cyclic
di-AMP (c-di-AMP) is involved in regulating sporulation, cell
size, and cell wall stress tolerance,3,4 and cyclic AMP-GMP (c-
AMP-GMP) has been implicated in affecting intestinal
colonization by bacteria.5 In addition, there is evidence that
these cyclic dinucleotides stimulate the innate immune
response in mammalian cells.6−8 Thus, cyclic dinucleotides
are of further interest as potential small molecule adjuvants for
vaccine development.9
As with other second messengers, cyclic dinucleotide
signaling appears to be tightly temporally and spatially
controlled. Thus, there is a critical need for in vivo biosensors
that can monitor their dynamics in order to understand how
physiological changes are signaled. A Forster resonance energy
̈
transfer (FRET)-based biosensor recently has been employed
to monitor c-di-GMP in Caulobacter crescentus.10 It exhibits a
modest decrease in net FRET signal upon ligand binding.
There are as yet no reported biosensors for c-di-AMP or c-
AMP-GMP.
The use of RNA as a tool for in vivo molecular sensing has
long been under-developed despite the natural ability of RNAs,
such as riboswitches, to selectively recognize small molecules.
Paige et al. recently showed that fluorescence turn-on of 3,5-
difluoro-4-hydroxybenzylidene (DFHBI) by the Spinach RNA
aptamer11 can be activated by ligands binding to aptamers
inserted within the Spinach sequence.12 Here we show that a
selective and sensitive fluorescent biosensor for c-di-GMP can
be generated by fusing a natural GEMM-I riboswitch aptamer
Figure 1. RNA-based fluorescent biosensors detect cyclic dinucleotide
second messengers. (a) Design scheme for a fluorescent biosensor that
detects cyclic dinucleotides. 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, and
Spinach stem loops are numbered in orange. (c) In vitro screen of Vc2-
Spinach biosensor variants for fluorescence activation in response to
cyclic dinucleotides. Error bars represent the standard deviation of
three independent experiments with duplicate samples.
Received: December 12, 2012
Published: March 14, 2013
© 2013 American Chemical Society
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Journal of the American Chemical Society
Communication
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 SEM for at least 50 cells. P-values from student’s t-test
comparisons for fluorescence changes discussed in text are shown.
wild-type (WT) Vc2-Spinach, exhibits selective fluorescence
activation in response to c-di-GMP (Figures 1c and S1). As
expected, the related RNA construct M1 Vc2-Spinach, which
harbors a disruptive mutation in the riboswitch aptamer
structure,13 exhibits very little fluorescence activation in
response to c-di-GMP (Figure 1b,c).
G20A Vc2-Spinach (Figures 3a and S5). Thus, the apparent
insensitivity of C92U and G20A/C92U biosensors is consistent
with comparable reductions in binding affinities leading to Kd
values much larger than the ligand concentration (100 μM)
used in the assay. Another WT Vc2-Spinach construct with an
artificial transducer stem in place of the second stem loop of
Spinach has been described that displays weak binding affinity
to c-di-GMP even at 25 °C.20 In this case, the difference in
affinity relative to our WT construct is likely due to alteration of
the Vc2 aptamer P1 stem sequence, which reduces ligand
binding.17
Since the concentration of c-di-GMP appears to range from
<50 nM to a few micromolar in bacteria,2 WT Vc2-Spinach
should be capable of detecting c-di-GMP at biologically
relevant concentrations. Furthermore, the dynamic range of
the RNA-based biosensor is larger than that of the previously
mentioned protein-based biosensor, which binds c-di-GMP
cooperatively as a dimer.10 The in vitro fluorescence signal of
WT Vc2-Spinach changes from 10% to 90% between 25 and
2000 nM c-di-GMP (Figure 2a). In comparison, the protein-
based biosensor exhibits similar affinity as the RNA biosensor,
but its FRET signal in vitro changes from 90% to 10% between
67 and 560 nM c-di-GMP.9 The RNA-based biosensor does
display relatively slow activation and deactivation rates (Figure
S6), so it would need to be improved if it is necessary to
monitor rapid dynamics.
We then tested the activity of WT Vc2-Spinach as a
fluorescent biosensor to detect c-di-GMP levels in live cells
(Figures 2b,c and S7). All RNA constructs were inserted into a
tRNA scaffold to improve stability upon expression in E. coli.21
The tRNA scaffold has a negligible effect on ligand binding
affinities (Figures S3 and S5). Plasmids encoding WT Vc2-
Spinach tRNA and variants of the diguanylate cyclase WspR
from Pseudomonas fluorescens were cotransformed into cells. We
verified that the expression levels of RNA constructs were
unaffected by expression of different enzymes (Figure S8).
Besides WT WspR,22 we analyzed the D70E mutant, which
mimics phosphorylation at that site and is constitutively active,
and the G249A mutant, which knocks out the conserved
GGDEF domain and is constitutively inactive.22 Thus, cells
expressing the WT biosensor and G249A WspR represent the
fluorescence signal for endogenous levels of c-di-GMP in E. coli.
As shown, the average fluorescence of these cells is 1.6-fold
We next tested whether specific mutations to the ligand
binding pocket would confer responsiveness to the other cyclic
dinucleotides of interest. The X-ray crystal structure of the Vc2
aptamer bound to c-di-GMP revealed that nucleobases C92 and
G20 (numbered using the riboswitch aptamer sequence, Figure
1b) form a Watson−Crick and Hoogsteen base pair,
respectively, with each of the guanine nucleotides of the
ligand.14,15 It has been shown that the single mutant C92U
binds c-AMP-GMP (Kd = 19
mutant G20A/C92U binds c-di-AMP (Kd = 1,200
1.7 nM)16 and the double
130
nM),14 albeit in each case nonselectively and with much poorer
affinity than the WT aptamer for c-di-GMP. The single mutant
G20A also has been shown to bind c-di-GMP (Kd = 0.21
0.07 nM),17 but its affinity for the other cyclic dinucleotides
was not determined. These three variants of Vc2-Spinach were
assayed for fluorescence activation by cyclic dinucleotides. It
was observed that G20A Vc2-Spinach responds robustly to c-di-
GMP and c-AMP-GMP but not to related compounds (Figure
S2), while the other variants exhibit little to no fluorescence
activation by any of the cyclic dinucleotides (Figure 1c).
Therefore, we focused the remainder of our analysis on the WT
and G20A Vc2-Spinach RNA constructs.
In order to model physiological conditions, in vitro
experiments were carried out at 37 °C in buffer containing 3
mM MgCl2, which is in the range of the estimated free Mg2+ in
the E. coli cytosol.18,19 Under these conditions, it was found
that WT Vc2-Spinach has an apparent Kd of 230 50 nM for c-
di-GMP (Figure 2a). This binding affinity is much poorer than
the previously reported value for the Vc2 aptamer alone and
can be attributed to two effects. First, WT Vc2-Spinach exhibits
increased sensitivity for c-di-GMP (Kd of 8 1 nM) at 25 °C,
10 mM MgCl2 (Figure S3), the conditions used in the previous
study.14 In parallel, greater fluorescence turn-on is observed
(Figure S4), consistent with stabilization of the RNA fold under
these conditions. The remaining difference in binding affinity
appears to be due to placement of the Vc2 aptamer within the
context of Spinach, as we observe the same affinity trends for
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Communication
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) 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 SEM for at least 50 cells. P-values from student’s t-test comparisons are shown.
above the nonspecific background, which is represented by cells
expressing Vc2 tRNA, a construct that does not contain the
Spinach aptamer (Figure S9). In comparison, cells expressing
the biosensor and WT or D70E WspR exhibit roughly the same
increases in average fluorescence (2.3- and 2.4-fold, respec-
tively) above the signal corresponding to endogenous levels.
These data are consistent with production of additional c-di-
GMP by the WT and constitutively active enzymes.
In contrast, cells expressing the M1 biosensor do not exhibit
average fluorescence above background except in the case
where D70E WspR is coexpressed. M1 Vc2-Spinach has very
weak binding to c-di-GMP, as we observed in vitro (Figure 1c),
so it appears that levels of c-di-GMP that are endogenous or
produced by WT WspR are below the threshold of detection.
However, D70E WspR produces c-di-GMP at extremely high
concentrations, estimated to be around 3 mM,23 and so elicits a
small fluorescence response from the mutant biosensor. For the
WT biosensor, the lack of a significant difference in
fluorescence with overexpression of the active WspR variants
suggests that each produces enough c-di-GMP to saturate the
biosensor and give maximal fluorescence signal. These data
demonstrate that WT Vc2-Spinach is a sensitive biosensor that
gives a measurable fluorescence response to endogenous c-di-
GMP and increased signal upon elevation of c-di-GMP
concentrations.
In order to analyze the G20A Vc2-Spinach aptamer for use in
vivo, its relative affinities for c-di-GMP and c-AMP-GMP were
determined. The G20A Vc2-Spinach aptamer has an apparent
Kd of 1000 150 nM for c-di-GMP and 4200 320 nM for c-
AMP-GMP at 37 °C, 3 mM MgCl2 (Figure 3a). The G20A
biosensor appears to be not sensitive enough to detect
endogenous c-di-GMP but, as expected, gives a fluorescence
signal upon expression of WT and D70E WspR (Figure S10).
In this case, the fluorescence signal with WT WspR is not
saturated and is clearly less than the D70E WspR signal. While
G20A Vc2-Spinach by itself cannot distinguish between c-di-
GMP and c-AMP-GMP, we considered that comparing the
ratio of fluorescence activation between the G20A and WT
biosensors may allow us to differentiate between responses to
the two cyclic dinucleotides.
D131A/D133A DncV mutant.6 However, it was not possible to
ascertain whether c-di-GMP also was being produced by this
enzyme in vivo. We found that cells expressing the WT
biosensor and D131A/D133A DncV have an average
fluorescence 1.6-fold above background, which is the same as
observed with inactive WspR and corresponds to detection of
endogenous c-di-GMP (Figure 3b,c). Expression of WT DncV
does not significantly change the average fluorescence of the
WT biosensor, providing direct evidence that this enzyme does
not produce c-di-GMP in vivo under these conditions.
Cells expressing the G20A biosensor and D131A/D133A
DncV exhibit an average fluorescence that is similar to
background, represented by M1 Vc2-Spinach (Figures 3b,c,
S11, and S12). This result agrees with G20A Vc2-Spinach
having a reduced sensitivity for c-di-GMP and E. coli cells not
making c-AMP-GMP. In contrast, cells expressing the G20A
biosensor and WT DncV exhibit greater than 4-fold increase in
fluorescence above background. Given the results from the WT
biosensor, this observation is consistent with fluorescence
activation of the G20A biosensor due to production of c-AMP-
GMP by WT DncV. Thus, we have engineered a biosensor that
can detect c-AMP-GMP, a newly discovered second messenger,
in live cells.
In summary, we have developed two different fluorescent
biosensors for live cell imaging of cyclic dinucleotides. We
demonstrate the ability to alter specificity of the RNA-based
biosensor by exploiting rational mutations to the ligand binding
pocket instead of by inserting distinct aptamers. Ongoing work
focuses on improving the binding characteristics of these first-
generation biosensors and on developing a fluorescent
biosensor for c-di-AMP. We aim to use our biosensors to
accurately quantitate changes in the intracellular levels of cyclic
dinucleotides upon different physiological stimuli, similar to
sensors for cGMP.24 It is envisioned that sensitive and specific
biosensors for each cyclic dinucleotide will help elucidate how
these different signals are integrated and transduced by bacteria.
ASSOCIATED CONTENT
■
S
* Supporting Information
Methods, supplementary experiments, and characterization of
c-AMP-GMP. This material is available free of charge via the
Internet at http://pubs.acs.org.
The dinucleotide cyclase DncV from V. cholerae was recently
shown to synthesize c-di-GMP, c-di-AMP, and c-AMP-GMP in
vitro, but it preferentially makes c-AMP-GMP in the presence
of all four nucleotide triphosphates.5 Also, mass spectrometry
analysis of cell lysates supports that c-AMP-GMP is produced
in vivo by WT DncV but not by the catalytically inactive
AUTHOR INFORMATION
■
Corresponding Author
mingch@berkeley.edu
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Journal of the American Chemical Society
Communication
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Daniel Cohen for technical assistance, Carl Onak for
discussion on microscopy, Jacob Vogan for assistance with
Northern blots, and the Chris Chang and Bertozzi laboratories
for instrument usage. This work is supported by an NIH New
Innovator Award (grant no. 1DP2OD008677) to M.C.H., an
NIH NRSA Training Grant in Chemical Biology (C.A.K.,
S.C.W.), and a Department of Defense NDSEG Fellowship to
C.A.K. M.C.H. holds a Career Award at the Scientific Interface
from the Burroughs Wellcome Fund.
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