Claims 32
From the paper Synthetic GPCRs for programmable sensing and control of cell behaviour · claims file · checked 2026-10-21: ready
- fig1c-antagonist-screen
Of 24 candidate dynorphin-derived antagonist peptides fused to the kappa-opioid DREADD, 16 auto-inhibited PAGER-TF, shifting the SalB EC50 more than tenfold.
Sixteen out of 24 peptides displayed antagonism in the context of PAGER TF by shifting the half-maximal effective concentration (EC 50 ) of the SalB response more than tenfold
Main text, paragraph on the antagonist peptide screen (after the Fig. 1 legend); Fig. 1c; Extended Data Fig. 1h - ed2-tev-reversible
All 16 antagonized constructs were reversibly antagonized; extracellular TEV protease cleavage re-sensitized PAGER-TF to SalB.
We found that all 16 constructs were reversibly antagonized
Main text, paragraph on the antagonist peptide screen; Extended Data Fig. 2 - fig1hi-gfp-relieves-inhibition
All anti-GFP PAGER-TF constructs responded, to varying degrees, to surface-expressed GFP on co-plated cells and to soluble GFP, with the shorter arodyn (1-6) giving the largest GFP response.
All anti-GFP PAGER TF constructs responded in varying degrees to both surface-expressed and soluble GFP ... we selected the shorter arodyn (1–6) peptide, which displayed sufficient antagonism and the greatest response to GFP
Main text, paragraph after the Extended Data Fig. 2 legend; Fig. 1h,i,k,l; Extended Data Fig. 2 - fig1op-linker-soluble-antigen
With flexible GGGS linkers between nanobody and antagonist, anti-GFP PAGER-TF is still activated by surface GFP but its response to soluble GFP is largely abolished.
when including GS linkers between the nanobody and peptide antagonist, anti-GFP PAGER TF could still be activated by surface GFP antigen ... but its response to soluble GFP antigen was largely abrogated
Main text, paragraph on the steric mechanism; Fig. 1o,p - ed3a-nanobody-swap
Swapping in other nanobodies gave functional receptors; nine anti-GFP and six anti-mCherry PAGER-TF constructs were strongly activated by their soluble antigen.
The remaining nine anti-GFP PAGER TF and all six anti-mCherry PAGER TF constructs showed strong activation in response to soluble recombinant GFP and mCherry, respectively
Results, PAGER can sense and respond to diverse antigens, paragraph 1; Extended Data Fig. 3a; Supplementary Fig. 4 - ed3b-detection-limits
The best anti-GFP PAGER-TF (LaG2) detects soluble GFP down to 1.5 nM and the best anti-mCherry PAGER-TF (LaM8) detects 100 nM mCherry.
The best anti-GFP PAGER TF (containing LaG2 nanobody) could detect soluble GFP down to 1.5 nM ... whereas the best anti-mCherry PAGER TF (containing LaM8 nanobody) could detect 100 nM mCherry protein
Results, PAGER can sense and respond to diverse antigens, paragraph 1; Extended Data Fig. 3b - ed3b-orthogonal
The anti-GFP and anti-mCherry PAGER-TF do not respond to each other's antigen.
Neither PAGER was responsive to the other fluorescent protein, illustrating the high specificity of these synthetic receptors
Results, PAGER can sense and respond to diverse antigens, paragraph 1; Extended Data Fig. 3b - ed3cd-reversibility-affinity
PAGER-TF with high-affinity nanobodies (LaG2, LaM6) remain activatable by SalB 30 min after antigen washout, while those with lower-affinity nanobodies (LaG17, LaM8) are unresponsive immediately after washout; reversibility is set by nanobody affinity.
PAGER TF containing high-affinity nanobodies such as LaG2 17 ( K d = 16–19 nM) or LaM6 17 ( K d = 0.26 nM) continued to be activated by SalB, even 30 min after antigen washout ... Thus the reversibility and ability of PAGER TF to function as a coincidence detector are largely controlled by the affinity of the nanobody
Results, PAGER can sense and respond to diverse antigens, paragraph 2; Extended Data Fig. 3c,d - fig2a-many-antigens
PAGER-TF built for 13 further antigens (VEGF, HGF, TNF, IL-17, IL-23, sIL-6R, CCL2, EGFR, HER2, CD38, PD-L1, SARS-CoV-2 spike, uPA) each respond to their cognate antigen and not to mismatched antigens.
In this way, we created PAGER TF for VEGF, HGF, TNF, IL-17, IL-23, soluble IL-6 receptor (sIL-6R), CCL2, EGFR, HER2, CD38, PD-L1, SARS-CoV-2 spike protein and urokinase-type plasminogen activator (uPA) ... Each PAGER was responsive to its cognate antigen, but non-responsive to mismatched antigens
Results, PAGER can sense and respond to diverse antigens, paragraph 3; Fig. 2a; Extended Data Fig. 5a - ed5bc-spike-nanobodies
Of five PAGER-TF made with different RBD-binding nanobodies, four responded to trimeric SARS-CoV-2 spike protein.
We made PAGER TF constructs using five different nanobodies that bind to RBD epitopes that are accessible in trimeric spike protein. All but one produced spike-responsive PAGER TF
Results, PAGER can sense and respond to diverse antigens, paragraph 4; Extended Data Fig. 5b,c - fig2c-macrophage-m1
PAGER-TF in HEK cells detecting VEGF, CCL2 or PD-L1 drives IFN-gamma secretion that converts co-cultured RAW 264.7 macrophages to an M1 state, with CD86 and NOS2 upregulated only with antigen and SalB.
Upregulation of the M1 markers CD86 and NOS2 was observed in antigen and SalB-treated samples, but not in negative controls
Results, PAGER can sense and respond to diverse antigens, paragraph 5; Fig. 2b-d; Extended Data Fig. 6 - fig2f-bite-killing
PAGER-TF sensing CCL2 or PD-L1 drives secretion of an anti-CD3/CD19 BiTE that increases killing of co-cultured Nalm6 tumour cells by primary T cells.
We used PAGER TF to sense the tumour antigens CCL2 23 or PD-L1 24 (or GFP as a control) and respond by secreting an anti-CD3/CD19 BiTE, which increased the killing of co-cultured CD19-expressing Nalm6 tumour cells by primary T cells
Results, PAGER can sense and respond to diverse antigens, paragraph 6; Fig. 2e,f - ed7-toxin-screen
Among muscarinic toxins, MT1 best gates the M1 DREADD and MT3 the M4 DREADD; none auto-inhibits the M3 DREADD.
We found that MT1 on M1 DREADD produced the greatest fold change in G q recruitment with TEVp treatment, indicating strong and reversible inhibition, whereas MT3 was the best toxin for gating M4 DREADD ... None of the MT proteins that we screened auto-inhibited the M3 DREADD
Results, Antigen-dependent G-protein activation via PAGER, paragraph 2; Extended Data Fig. 7c-e; Supplementary Fig. 6 - fig3el-pagerg-coupling
PAGER-Gq, -Gs, -Gi and -G12 couple antigen and drug to their G-protein partners, shown by antigen- and CNO-dependent ERK phosphorylation and antigen- and DCZ-dependent TRUPATH activation.
First, we used western blotting to show antigen and CNO-dependent stimulation of ERK phosphorylation ... Second, we used the TRUPATH assay to confirm antigen and DCZ-dependent activation of the corresponding G-protein partner for each PAGER G
Results, Antigen-dependent G-protein activation via PAGER, paragraph 3; Fig. 3e-l; Extended Data Fig. 8c-e - fig3mo-second-messengers
Anti-mCherry PAGER-Gq raises cytosolic Ca2+, and PAGER-Gs and PAGER-Gi raise and lower cAMP, in an mCherry- and DCZ-dependent way.
We also used the Ca 2+ indicator GCaMP6s 32 to image mCherry and DCZ-dependent increases in cytosolic Ca 2+ driven by anti-mCherry PAGER Gq ... we could observe mCherry-dependent increases ... or decreases ... in cAMP
Results, Antigen-dependent G-protein activation via PAGER, paragraphs 3 and 4; Fig. 3m-o; Extended Data Fig. 9 - fig3qs-tnf-vegf-sensitivity
Anti-TNF PAGER-Gq responds to TNF down to 2 nM and anti-VEGF PAGER-Gq detects 0.2 nM VEGF.
Our dose titration showed that anti-TNF PAGER Gq could respond to TNF concentrations as low as 2 nM ... Anti-VEGF PAGER Gq could detect 0.2 nM VEGF
Results, Antigen-dependent G-protein activation via PAGER, paragraph 4; Fig. 3p-s - fig4bd-culture-calcium-suppressed
In cultured rat cortical neurons expressing anti-mCherry PAGER-Gi, soluble or cell-surface mCherry with CNO or DCZ strongly suppresses calcium activity within seconds, while drug alone does not.
Untreated neurons and CNO or DCZ-only treated neurons exhibited transient fluctuations in basal calcium ... However, addition of soluble mCherry along with CNO or DCZ strongly suppressed calcium activity within seconds, as did co-culturing with HEK cells that express cell-surface mCherry
Results, Customized cell behaviours driven by PAGER G, paragraph 2; Fig. 4b-d; Extended Data Fig. 11d-f - methods-slice-sample
The slice data comprise 15 cells from 7 mice recorded with DCZ alone and 26 cells from 6 mice recorded with DCZ plus mCherry.
Data were collected from n = 15 cells, 7 mice with DCZ, n = 26 cell, 6 mice DCZ + mCherry
Methods, Neuronal electrophysiology assay in brain slices, Slice electrophysiology; Fig. 4i,j legendHow it will be judged
Counts of cells and animals are exact, so the tolerance is zero. The paper does not say whether the counts are taken before or after its exclusion of cells with initial Rin above 500 megaohms (Methods). The readings are (a) all cells per condition in the dataset; (b) the cells per condition that pass that exclusion, with initial Rin taken from the first sweep of the recording, Rin as defined in fig4i-rmp. Reproduced when all four counts match under one reading; partially reproduced when the cell counts match and the mouse counts do not, or the reverse. Any recording that appears twice is counted once.
- methods-mcherry-preincubation
Every cell recorded with DCZ plus mCherry was preincubated in 1 uM mCherry for 30 to 150 min, 73.4 min on average.
All cells recorded with DCZ + mCherry also underwent a prior preincubation in mCherry (1 µM; range: 30–150 min, mean: 73.4 min)
Methods, Neuronal electrophysiology assay in brain slices, Slice electrophysiologyHow it will be judged
From the preincubation duration the dataset records for each DCZ plus mCherry cell. Reproduced when the mean is within tolerance and every duration lies within 27 to 165 min (the range widened by 10% of each bound). The paper does not say whether the mean is over all recorded cells or over the 26 analysed; both are shown and either matching suffices.
- fig4i-rmp-hyperpolarization
In acute hippocampal slices, PAGER-Gi-expressing CA1 neurons show a greater hyperpolarizing shift in resting membrane potential with 100 nM DCZ plus 1 uM mCherry than with DCZ alone (mixed linear model, Z = 3.17, P = 0.0015).
Neurons displayed a greater hyperpolarizing shift in RMP when presented with DCZ plus mCherry than with DCZ alone
Results, Customized cell behaviours driven by PAGER G, paragraph 3; Fig. 4i and legendHow it will be judged
Measures from the Methods (Slice electrophysiology). Per sweep, RMP is the mean membrane potential over the 500 ms before any current injection. Rin is the steady-state voltage change to the -100 pA, 200-ms step (step voltage minus that sweep's RMP) divided by -100 pA; the steady-state window is not given, and the readings are the last 50 ms and the last 100 ms of the step. Cells whose initial Rin (first sweep) exceeds 500 megaohms are excluded (the paper prints "mΩ"), and sweeps with RMP above -50 mV or with no action potential during the ramp. Action potential detection is not given; the readings are (A1) an upward crossing of 0 mV and (A2) dV/dt above 20 V/s. The change "upon 2-min DCZ addition" is not defined beyond "before, during and after" the application; the readings are (W1) mean over included sweeps from DCZ onset to 2 min after onset minus the mean over included sweeps before onset, and (W2) mean over included sweeps from onset to the end of the recording minus the same baseline. Onset is the application time the dataset records. The test is a mixed linear model (statsmodels, as the Methods name) with treatment as fixed effect and a random intercept per mouse (Methods, Statistical analysis); the unit is not given, and the readings are (M1) one change per cell and (M2) every included sweep's change from baseline, with cell as a variance component within mouse. Under each combination of readings, the result is reproduced when the DCZ plus mCherry change is more negative than the DCZ change, the treatment effect has p < 0.05, and |Z| is within tolerance; partially reproduced when direction and p < 0.05 hold but |Z| is outside tolerance; not reproduced otherwise. The outcome is the one all combinations give; if they differ, the claim is blocked as methods_underspecified with every result shown. Group means and s.e.m. of the change are reported, as the figure plots them.
- fig4j-rheobase-increase
The rheobase current needed to evoke action potentials increases more with DCZ plus mCherry than with DCZ alone (mixed linear model, Z = 2.60, P = 0.0093).
Similarly, the rheobase current needed to evoke action potentials increased in DCZ plus mCherry neurons, compared with DCZ-only samples
Results, Customized cell behaviours driven by PAGER G, paragraph 3; Fig. 4j and legendHow it will be judged
Rheobase per sweep is the current being delivered when the first action potential of the ramp (-150 to +500 pA over 2 s) is evoked (Methods), read from the recorded stimulus at the time of detection; the ramp formula is shown as a check. Whether the holding current is included is not stated; it cancels in a change if constant, and the author shows whether it is. Exclusions, action potential detection (A1, A2), change windows (W1, W2), the mixed model (M1, M2) and the Rin windows used for the cell exclusion are as in fig4i-rmp. Under each combination, reproduced when the DCZ plus mCherry rheobase change is larger than the DCZ change, p < 0.05, and |Z| within tolerance; partially reproduced when direction and p < 0.05 hold but |Z| is outside tolerance; not reproduced otherwise. The outcome is the one all combinations give; if they differ, blocked as methods_underspecified with every result shown.
- ed12b-input-resistance-trend
Input resistance tends to decrease more with DCZ plus mCherry than with DCZ alone, a trend that does not reach significance (mixed linear model, Z = 1.84, P = 0.0653).
We also observed a similar time course trend for decreasing input resistance in DCZ plus mCherry neurons, consistent with hM4Di-mediated inhibition
Results, Customized cell behaviours driven by PAGER G, paragraph 3; Extended Data Fig. 12b and legendHow it will be judged
Rin, exclusions, change windows (W1, W2), Rin steady-state windows, action potential detection (A1, A2) and the mixed model (M1, M2) as in fig4i-rmp. Under each combination, reproduced when the DCZ plus mCherry Rin change is more negative than the DCZ change and |Z| is within tolerance; partially reproduced when the direction holds and |Z| is outside tolerance; not reproduced when the direction is reversed. The p-value is reported either way. The outcome is the one all combinations give; if they differ, blocked as methods_underspecified with every result shown.
- ed12cd-expression-baseline
PAGER-Gi expression level (mEGFP intensity) is not correlated with baseline rheobase or input resistance, in either group, including in the presence of soluble mCherry.
The degree of mEGFP expression, as an indicator of PAGER Gi expression levels in neurons, supports PAGER Gi alone having no impact on baseline rheobase (c) or input resistance (d) properties, including in the presence of soluble mCherry
Extended Data Fig. 12c,d,h legend; Results, Customized cell behaviours driven by PAGER G, paragraph 3How it will be judged
A claim of absence. The paper's test is a Pearson correlation (Extended Data Fig. 12 legend) between each cell's mEGFP mean intensity, measured in a freehand soma ROI of a widefield image taken before the recording, and its baseline value, within each group. The paper does not define the baseline; the readings are (B1) the mean over included sweeps before DCZ onset and (B2) the first included sweep. Rin, the sweep and cell exclusions and their readings (Rin windows, A1, A2) are as in fig4i-rmp. The threshold is P < 0.05, the paper's significance level (Methods, Quantification and Data Analysis). Under each combination, reproduced when no correlation (rheobase and Rin, each group) has P < 0.05; not reproduced when any does; r and P are reported for all four. If the combinations disagree, blocked as methods_underspecified with every result shown. The ROIs are freehand and not published, so the test uses the per-cell intensities or ROIs the dataset holds.
- ed12ef-expression-shift
PAGER-Gi expression level correlates with the size of the rheobase and input resistance shifts after DCZ plus mCherry, but not after DCZ alone.
Construct expression levels were correlated with the degree of rheobase (e) and input resistance (f) shifts following DCZ+mCherry exposure, but not with DCZ alone
Extended Data Fig. 12e,f,h legendHow it will be judged
Pearson correlation of each cell's mEGFP mean intensity (as in ed12cd) with its rheobase change and its Rin change, defined and computed under the readings of fig4i-rmp (W1, W2; A1, A2; Rin windows), separately per group, at P < 0.05 (Methods, Quantification and Data Analysis). The paper does not give the sign; it is reported. Under each combination, reproduced when both correlations have P < 0.05 in the DCZ plus mCherry group and neither in the DCZ group; partially reproduced when this holds for one of the two measures; not reproduced otherwise. The outcome is the one all combinations give; if they differ, blocked as methods_underspecified with every result shown.
- ed12g-expression-rmp
Shifts in resting membrane potential are not specifically linked to PAGER-Gi expression level.
Resting membrane potential (RMP) shifts were not specifically linked to mEGFP expression levels (g)
Extended Data Fig. 12g,h legendHow it will be judged
A claim of absence, on the paper's test. Pearson correlation of each cell's mEGFP mean intensity (as in ed12cd) with its RMP change (fig4i-rmp, readings W1 and W2, with the sweep and cell exclusions and readings A1, A2 and the Rin windows of fig4i-rmp), in each group, at P < 0.05. The legend's "not specifically linked" allows two readings, and its table (Extended Data Fig. 12h) could not be read from the text. (S1) No link in either group; reproduced when neither group's correlation has P < 0.05, not reproduced when one does. (S2) No link specific to DCZ plus mCherry, set against the pattern of panels e and f; reproduced unless the DCZ plus mCherry correlation has P < 0.05 and the DCZ correlation does not. The claim is reproduced when it holds under S1 or S2, and the notebook says which. r and P are reported for every reading; if the window and detection readings disagree, blocked as methods_underspecified.
- fig4lm-t-cell-chemotaxis
Primary human T cells expressing anti-mCherry PAGER-Gi migrate towards mCherry in the presence of DCZ, but not towards GFP, and pertussis toxin blocks this migration.
migrate towards mCherry in the presence of DCZ, but not towards a non-cognate antigen (GFP) ... Inhibition of G i activation using pertussis toxin blocked PAGER-mediated migration
Results, Customized cell behaviours driven by PAGER G, paragraph 4; Fig. 4k-m (the text cites the panels as Fig. 4l,m and the expression histogram as Extended Data Fig. 12h, which the legends label Fig. 4k,l and Extended Data Fig. 12i) - fig4mn-chemotaxis-kinetics-dose
PAGER-mediated T cell migration is detectable within minutes of antigen exposure, and 100 nM mCherry suffices to stimulate it.
we could detect PAGER-mediated migration within minutes of antigen exposure ... and as little as 100 nM mCherry was needed to stimulate migration
Results, Customized cell behaviours driven by PAGER G, paragraph 4; Fig. 4m,n (cited in the text as Fig. 4n,o) - ed13-dcz-sensor
A GRAB-type sensor from human M4 (hM4-1.0) responds to acetylcholine with about a twofold maximal response and an apparent affinity near 225 nM; with DREADD mutations it becomes DCZ1.0, with a 1.7-fold fluorescence increase to DCZ and no coupling to Gi.
obtained an acetylcholine (ACh) sensor, hM4-1.0, with good membrane trafficking, a maximal response of around twofold, and an apparent affinity of approximately 225 nM ... producing DCZ1.0, which resulted in 1.7-fold fluorescence turn-on in response to the DREADD ligand DCZ ... We confirmed that this sensor does not couple with downstream G i protein, unlike hM4Di
Results, Real-time fluorescent sensors based on PAGER, paragraph 1; Extended Data Fig. 13c-k - fig5d-mcherry-pagerfl
Anti-mCherry PAGER-FL is sensitized 300-fold to DCZ by mCherry but not by BFP.
Fluorescence measurements showed 300-fold sensitization to DCZ in the presence of mCherry but not non-cognate antigen (BFP)
Results, Real-time fluorescent sensors based on PAGER, paragraph 2; Fig. 5c-e - fig5g-bfp-pagerfl
Anti-BFP PAGER-FL (LaG2 nanobody) is sensitized 130-fold to DCZ by BFP but not by mCherry.
Anti-BFP PAGER FL , generated by replacing LaM6 with the GFP- and BFP-binding LaG2 nanobody, showed 130-fold sensitization to DCZ in the presence of BFP but not mCherry
Results, Real-time fluorescent sensors based on PAGER, paragraph 2; Fig. 5f-h - fig5jl-pagerfl-kinetics
mCherry binds anti-mCherry PAGER-FL rapidly (t50 24 s), while the green fluorescence response is slower (t50 3.1 min).
Whereas binding of mCherry to anti-mCherry PAGER FL was rapid (time constant ( t 50 ) = 24 s), the green fluorescence response of PAGER FL was slower, with a t 50 of 3.1 min
Results, Real-time fluorescent sensors based on PAGER, paragraph 2; Fig. 5i-l - fig5mp-tnf-vegf-pagerfl
Anti-TNF and anti-VEGF PAGER-FL respond to their antigens in the presence of DCZ, with response size and kinetics similar to anti-mCherry PAGER-FL.
the application of 1 μM TNF elicited fluorescence increase in the presence of DCZ ... The response magnitude (Δ F / F 0 ) and kinetics of anti-TNF PAGER FL and anti-VEGF PAGER FL were similar to those of anti-mCherry PAGER FL
Results, Real-time fluorescent sensors based on PAGER, paragraph 3; Fig. 5m-p; Extended Data Fig. 13l,m
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