Peer review of: A STING-based biosensor affords broad cyclic dinucleotide detection within single living (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Pollock, Zaver, and Woodward developed a genetically-encoded FRET sensor of cyclic dinucleotides 
based on the mouse STING binding domain. The authors characterize their sensor, BioSTING, with 
studies using purified protein, lysates, and live HEK cells. They demonstrate applications of 
BioSTING in several promising in vitro formats for both endpoint and time course experiments, 
including flow cytometry and imaging applications. Overall, the manuscript is generally well written 
and the results are clear and support their conclusions for the most part, with a few major and 
minor comments that need clarification: 
 
Major Points: 
1) BioSTING has a high affinity for cGAMP of ~50nM, which the authors state matches 
physiologically relevant concentrations. There is a major concern that the sensor itself may still 
interfere with signaling by buffering fluctuations in cGAMP levels similar to the concern faced with 
calcium sensors. Expression levels of genetically encoded sensors may range from 0.1 to 1 uM for 
bright expression. With the sensor’s high affinity and slow dissociation kinetics this could bind up 
cGAS-produced cGAMP after cell activation to alter the kinetics or attenuate the amplitude 
downstream signaling. The authors need to test that this is not happening, perhaps by IRF 
dimerization or IFN expression experiments with cells expressing the STING pathway that have 
been transfected with and without sensor and mutant control sensor. 
 
2) BioSTING is proposed to operate via intramolecular FRET within the one monomer that exhibits 
a conformational change, while the other monomer does not. However, BioSTING still relies on 
dimerization for nucleotide binding. Is there any evidence of heterodimerization between a 
BioSTING monomer and endogenous STING? If this were to occur, sensor expression could again 
affect downstream signaling. Perhaps the authors could comment if there is any evidence of ER 
localization of BioSTING in a STING expressing cell type, which might suggest interactions with 
endogenous STING. Or maybe do a pulldown experiment? 
 
3) For the discussion of Fig. 2 and BioSTING’s application to quantification in cell extracts, there is 
still a concern that other cellular factors could alter the sensor’s performance and skew 
quantification. The authors could compare the sensor’s dose responses for purified protein and 
CDNs in chemically defined solutions (as in Fig. 1) versus doped into untransfected cell lysates. 
 
4) What is the variance in the baseline absolute FRET ratios when BioSTING is expressed in 
unstimulated cells? A FRET dynamic range of 20% is decent for a first-generation sensor in which 
the authors have done so much to admirably show that it is useful. However, it poses a concern 
with quantification of cGAMP concentrations because this relies on a standard curve based on a 
logarithmic concentration scale that is prone to large errors. From the flow cytometry data in Fig. 
S2e it looks like the variance is large relative to the maximum FRET change, so it would be 
appropriate to discuss the need for if not demonstrate a way to calibrate the responses, at least 
perhaps with a maximal stimulation point. 
 
5) Overall the explanation are very clear, with the one exception being the discussion of 
intracellular cGAMP measurement in Fig.3 and Fig. S2 and the Discussion. The authors compare 
empty vector control to cGAS over expression. With cGAS overexpression they estimate 
micromolar concentrations of cGAMP, but there needs to be more explicit explanation of where 
estimates of “physiologically relevant” cGAMP concentrations are coming from because 
heterologous expression of cGAS in HEK cells is the only system used. Similar to point (1) above, 
at minimum there should be some test with maybe an immune cell line with intact STING 
signaling, even if with just extracts tested in parallel with BioSTING and EIA quantification. 
 
 

Minor Points: 
1) Fig.3’s Legend appears to have gotten jumbled a bit with (a) and (c) reversed. 
2) It would be helpful to give some rationale for the choice of the mTFP-mKO FRET pair in the first 
results section where the engineering is described. It seems like it was chosen with the intent for 
flow cytometry applications, but is that true? Were there other reasons that might be of interest 
for others looking to design FRET sensors? 
3) Is there any FRET ratio drift when the sensor (or the control mutant) are expressed by itself in 
unactivated cells? Difference in maturation time could cause drift in basal FRET ration signals that 
would through off quantitation for longer time course experiments. 
 
Comment: 
Related to point (2) above - Do the authors think that the asymmetry between monomer 
conformational dynamics is true for a functional BioSTING dimer? If only one monomer is actually 
undergoing the conformational change, then the other monomer is reducing BioSTING’s practical 
dynamic range because your signal change is sitting on top of a high baseline fluorescence and 
you would only every expect half of your molecules to exhibit a FRET change. Is there any way to 
define an asymmetrical tandem dimer in which the dynamic monomer is fused to the FPs and the 
static monomer is bare? 
 
 
 
Reviewer #2: 
None 
 
Reviewer #3: 
Remarks to the Author: 
Detection methods of CDNs are limited, and there is an urgent need for a detection method of 
real-time monitoring of CDN synthase activity and inhibition. A recent paper by Mardjuki et al 
reported an assay that can quantify cGAMP in solution and in cell lysates. Compared to the 
Mardjuki method, BioSTING is the first method that can measure cGAMP concentrations in live 
cells and, thus, provides a major advance to the field. However, the ability of this tool to quantify 
cGAMP accurately in vitro has not been demonstrated in an as robust manner as Mardjuki’s 
method. The author should either tune down the language and acknowledge this or perform a few 
key experiments. 
Major comments: 
1. Can the authors determine the range of quantification of BioSTING? This is significant because 
LC-MS/M and the Mardjuki method are robustly quantitative and EIA can be quantitative when 
cGAMP is diluted into its dynamic range. For example, in Figure 1E – Why is there a large jump in 
signal between 125 and 250 ng of plasmid? Does this speak to the lack of dynamic range of the 
sensor (it underreports at low concentration and overreports at higher concentrations) or is this 
actually reflective of how much intracellular cGAMP there is? Can cGAMP be quantified here using 
another method? It is important to demonstrate the quantitative powers of BioSTING or the author 
can remove such claims. 
2. The authors note that the FRET maximum is 20% deltaR/R0. Can the authors clearly state the 
sensor’s range, max and min in terms of deltaR/R0 and the corresponding cGAMP concentration? 
3. The IC50 is shifted higher in Fig 3d (nucleofecting cGAMP into cells) compared to Fig 1d (in 
vitro). Is this a short coming of BioSTING or a limitation of nucleofection? 
4. Line 230. Do authors always have to measure with EIA/another method in order to quantify, or 
can you do a standard curve in cells? Figure 3D has no points below 3 μM or so, besides the 0 nM 
cGAMP point. Would like to see points below this in the standard curve to see how well the 
standard curve can actually quantify intracellular cGAMP, because without this data it is not valid 
to then conclude that there is 5-50 nM cGAMP in cells (Line 234). 
5. A c-di-GMP BRET sensor was reported by Dippel et al. Is BioSTING better in someway? 
6. Given that the single mutants Y239S T262A and R231H don't appear to affect 3'3'-c-di-GMP 
binding, it's surprising that the triple mutant has reduced FRET signal. As such, it is unclear if the 

triple mutant is a weaker 3'3'-c-di-GMP binder or if the reduced FRET signal is unrelated to 3'3'-c-
di-GMP binding. Can the authors test the triple mutant's affinity for 3'3'-c-di-GMP in vitro? 
Alternatively, can they show that the triple mutant is capable of reaching the same max FRET 
response as WT BioSTING? 
7. The authors have used HEK 293Ts as a model cell line, which do not express cGAS or STING 
endogenously. Can they demonstrate use of BioSTING in cell lines that expresses endogenous 
levels of cGAS (and stimulate with DNA)? In addition, can they demonstrate use in a cell line that 
already expresses STING? Will competition between endogenous STING and BioSTING for cGAMP 
affect the detection of cGAMP by BioSTING? 
8. Does expression level of BioSTING affect FRET signal for experiments that involve 
transfected/electroporated/extracellular cGAMP? Given the slow dissociation rate of cGAMP from 
STING demonstrated in Figure 1g, it's likely that BioSTING sequesters cGAMP, and that higher 
BioSTING levels will result in artificially higher intracellular cGAMP levels. The authors should 
perform a doxycycline titration in their BioSTING HEK293T cells and evaluate how that affects 
FRET signal in response to a constant concentration of extracellular cGAMP. 
9. Figure 5b should be accompanied by a Western blot to show that the levels of expressed Poxin 
are indeed constant across the cGAS titration (minor point). 
 
Typos: 
1. Line 111: STING isoforms should be referred to as STING alleles. (isoforms typically refer to 
different proteins originating from the same gene as a result of alternative splicing). 
2. Line 117: double that (that, ..., that). 
3. Line 214: Should be 2'3'-cGAMP, not 2',3'-cGAMP. 
4. Figure 1E: Please add what concentration of nucleotide was used to the caption. 
5. Figure 1F: Please add how much excess nucleotide is present. 
6. Figure 1G: Please add how long was the interval between the addition of hot cGAMP was the 
addition of cold cGAMP. What concentrations of CDNs were used? 
7. Figure S2D: Assuming 100% efficiency of transfection, what concentrations of cGAMP would end 
up being in the cells. The amount of cGAMP in ng is not helpful as a reference point, and the x-axis 
in 3D is given as concentrations of cGAMP. 
8. Figure 4E: Please add how much of each plasmids was transfected. 
9. Figure legends for 3A and 3C are mixed up. 
10. Figure legends for 3G and 3H are mixed up.