Design and Characterization of the RAS Sensor.

(a) Design of the binding component of the RAS sensor. Inspired by natural RAF (left), the RAS-binding component of the sensor (right) comprises RAS binding domain (RBDCRD), a linker, and a NarX-derived transphosphorylation domain. (b) Schematic of the RAS sensor composition and mechanism of action. The sensor’s genetic payload is encoded on four plasmids. Two plasmids express the RAS-binding components: RBDCRD fused, respectively, to NarXN509A or NarXH399Q. The third plasmid expresses NarL, and the fourth plasmid encodes the output protein (mCerulean) under the control of a NarL response element (NarL-RE) in front of a minimal promoter (TATA). Upon RAS activation, the RBDCRD domain of the RAS-binding components bind to RAS-GTP. This binding leads to a forced dimerization of the NarX domains and a transphosphorylation of NarXN509A, in turn phorsphorylating NarL. Phosphorylated NarL binds its response element on the output plasmid, inducing the expression of the output protein. (c) Sensor activation by mutated RAS. The bar chart shows output expression in HEK293 cells co-transfected with the RAS Sensor and either KRASG12D or KRASWT. (d) Dose-response curve and dependence of the RAS sensor on functional RAS binding. Output expression of RAS sensors with either RBDCRD wild-type (blue) or RBDCRD with R89L (red), C168S (green), or both (purple) mutations. The dashed line represents conditions where the NarX-fusion plasmids were replaced with a non-coding plasmid (control). (e) Dependence of sensor output on RAS levels. Output expression of the RAS sensors measured with increasing amounts of KRASG12D (blue), KRASWT (orange), or negative control (black) plasmid. (f) Input-output curve. Correlation of the output expression with the RAS-GTP levels in HEK293 cells, measured by a luminescence RAS-pulldown ELISA assay. To alter RAS-GTP levels, the cells were transfected with different amounts of either KRASG12D (blue), KRASWT (orange), or KRASWT + Sos-1 (purple) plasmids. Pearson’s correlation is shown as R2. mCerulean output expression was measured by flow cytometry and normalized to a constitutively expressed mCherry transfection control. Mean values were calculated from biological triplicates. Error bars represent +/-SD. Significance was tested using an unpaired two-tailed Student’s t-test. ****p < 0.0001.

Mechanism of Action.

Effect of differential RAS activation on the steps considered necessary for RAS Sensor activation. RAS activation in HEK293 cells was manipulated by co-expressing KRASG12D, KRASWT, Sos-1 (a guanine nucleotide exchange factor that activates endogenous RAS), or NF1 (a GTPase-activating protein that deactivates endogenous RAS). In the control condition the cells are transfected with a non-coding plasmid, here it represents the endogenous RAS activation. Schematics on top of the graphs illustrate how and what part of the Mechanism was investigated. (a) Expression levels of the RBDCRD-NarX-SYFP2 fusion protein measured by flow cytometry in the presence of various KRAS modulators (x axis labels). (b) RAS binding of the RBDCRD-NarX-SYFP2 fusion protein approximated by calculating the ratio of membrane to total SYFP2 signal for each cell. Intracellular localization of SYFP2 was measured using confocal microscopy. The micrographs below show representative images for each condition. Scale bars = 50 µm. (c) Dimerization of the NarX fusion proteins assessed by transfecting two complementary RBDCRD-NarX-split.mVenus fusions and measuring the mVenus fluorescence by flow cytometry. (d) Output expression after transfection with the full RAS Sensor measured by flow cytometry. In a, c & d the fluorescent signals were normalized to a constitutively expressed transfection control. Each symbol represents one biological replicate (a: n=9, c-d: n=3). The error bars represent +/-SD. In b the fluorescence at the membrane was normalized to the total fluorescence for each cell. Violin plots in b represent 560 (KRASG12D), 322 (KRASWT), 482 (Control), 226 (NF1), or 1194 (Sos-1) cells from three biological replicates. Significance was tested using an ordinary one-way ANOVA with Dunnett’s multiple comparisons to compare each condition with the control condition (endogenous RAS activation). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Tunability of the RAS sensor.

(a) 3D structure of the RAS Sensor dimerizing at the membrane. The structure of the RBDCRD-NarX fusion proteins (orange and yellow) was predicted using AlphaFold and aligned with existing NMR structures of RBDCRD (blue) bound to a KRAS-dimer (green) at the membrane. The ATP-binding site N509 (purple) and the phosphorylation site H399 (pink) are highlighted as spheres. Onthe top RBDCRD and NarX are fused with a 1x GGGGS linker and on the bottom with a 6x GGGGS linker. (b) Effect of different linkers in the RBDCRD-NarX fusion protein of the RAS Sensor. The heatmaps show the output expression in HEK293 co-transfected with KRASG12D when using different numbers of repeats of a flexible (GGGGS) or a rigid (EAAAK) linker in the RBDCRD-NarX fusion proteins. The graph above shows the corresponding fold change between output expression in cells with KRASG12D and KRASWT. (c) Effect of different binding domains (BD) fused to NarX in the RAS Sensor. The heatmap shows the output expression in HEK293 co-transfected with 15 ng/well of KRASG12D (blue), KRASWT (orange), or NF1 (green), a GTPase-activating protein that deactivates endogenous RAS. The bars above show the corresponding fold changes between cells with KRASG12D and KRASWT (black) or KRASG12D and NF1 (green). (d) Effect of different transactivation domains (TAD) fused to NarL in the RAS Sensor. The heatmap shows the output expression in HEK293 co-transfected with KRASG12D (blue), KRASWT (yellow) or NF1 (green). The bars above show the corresponding fold changes between cells with KRASG12D and KRASWT (black) or KRASG12D and NF1 (green). mCerulean output expression was measured using flow cytometry and normalized to a constitutively expressed mCherry transfection control. Mean values were calculated from three (b) or two (c-d) biological replicates. Error bars were calculated using error propagation rules.

Design of Multi-Input RAS-targeting Circuits.

(a) Schematic of the RAS-targeting circuit with an AND-gate between mitogen-activated protein kinase (MAPK) sensors and the direct RAS Sensor. Dimerization of RAS activates the MAPK pathway and its downstream transcription factors. These transcription factors bind the synthetic response elements (RE), expressing the parts of the RAS Sensor. The RBDCRD-NarX proteins then bind activated RAS, dimerize, and propagate the signal to NarL, leading to output expression. The logic diagram of the resulting coherent feed-forward loop with AND-gate logic is shown in the dotted box. (b) Schematic of the transcription factor binding sites present in the response elements. Multiple repeats of the binding sites were placed upstream of a minimal promoter (TATA) driving expression of the RAS Sensor parts. (c) Expression levels with the MAPK response elements. The heatmap shows the direct expression of mScarlet of the different REs in HEK293 cells co-transfected with 15 ng/well of either KRASG12D (blue), KRASWT (orange), or NF1, a protein that deactivates endogenous RAS (green). The bars above show the corresponding fold changes between cells with KRASG12D and KRASWT (black) or KRASG12D and NF1 (green). (d) RAS-dependency of the SRE-, pFos-, and PY2-response elements. Direct mScarlet expression of the response elements in HEK293 cells co-transfected with different amounts of KRASG12D (blue), KRASWT (orange), or non-coding control plasmids (red). (e) RAS-dependency of the Elk-RE when additionally overexpressing Elk-1. RAS titration as described in d. (f) Dynamic range of the RAS-targeting circuits. Fold change in mCerulean output expression between HEK293 co-transfected with 1.67 ng/well of KRASG12D and KRASWT. In the RAS targeting circuits, NarL-VP48 and/or the RBDCRD-6xfL-NarX fusion proteins were expressed using different MAPK-REs or a constitutive promoter (EF1a). Fluorescent protein expression was measured by flow cytometry and normalized to a constitutively expressed transfection control. Mean values were calculated from two (c-e) or three (f) biological replicates. PY2: polyoma virus enhancer domain; SRE: Serum response element; pFos: minimal promoter of c-fos; AP1: activator protein 1; Elk: Ets-like protein; Myc: myelocytomatosis protein. Detailed response element design is shown in Supplementary Fig. 4.

Modularity of the RAS-targeting Circuits.

(a) Screening of RAS circuit variants with different parts. mCerulean output expression in HEK293 co-transfected with 1.67 ng/well of KRASG12D versus KRASWT representing the ON-versus OFF-state of the screened circuits. The gray shading of the symbols represents the dynamic range (fold change between ON and OFF). Circuits with a high dynamic range (> 100+/-SD) are highlighted. The pie chart shows the prevalence of the response elements (RE) among the hits. (b) Prevalence of the circuit parts among the hits. On the left for all circuits where the RE expressed NarL (RE_NarL) and on the right where the RE expressed the NarX proteins (RE_NarX). TAD: transactivation domain, BD: binding domain. RA: Ras association domain of Rassf5 (c) Effect of different circuit parts on the output expression in HEK293 with KRASG12D (blue) and KRASWT (orange) fitted using a generalized linear regression. The EF1a expressed RAS Sensor with RBDCRD, a 6x flexible linker in the NarX fusion proteins, and VP48 as TAD fused to NarL was set as the reference sensor. The graph shows the model coefficients, which can be interpreted as the effect on output expression when a part in the reference sensor is replaced by the part indicated on the x-axis. (d) Expression of all circuit parts via response elements. Fluorescence histograms of mCerulean-positive cells comparing ON-(KRASG12D) and OFF-state (KRASWT) of RAS circuits when either NarL (blue), the NarX proteins (violet), or all parts (red) are expressed via REs. The tested circuits contain RBDCRD as BD, a 6x flexible linker and F.L.T. as TAD, the parts that led to the hits with the highest dynamic range. The table below shows the parts used in each of the tested circuit. (e) Microscopy images showing the mCerulean expression of the conditions from d. mCerulean output expression was measured by flow cytometry and normalized to a constitutively expressed mCherry transfection control. Each circuit was measured in three biological replicates. Error bars represent +/-SD. Significance in c was tested using the Wald test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Translation into Cancer Cells.

(a) RAS Sensor activation in colon cancer cells. Microscopy images of the mCerulean output expression in HCT-116 wild-type cells harboring a homozygous KRASG13D mutation (HCT-116WT; top row) and HCT-116 KRAS knock-out cells (HCT-116k.o.; bottom row) transfected with the initial RAS Sensor (right) or a RAS Sensor with an R89L mutation in the Ras binding domain (left). (b-d) Effect of different circuit parts in colon cancer cells. Boxplots of dynamic range of different RAS-targeting circuits grouped by to the circuit parts of interest they contain. The circuit parts investigated were: the response elements in b, the binding domain fused to the NarX proteins in c, and the transactivation domain fused to NarL in d. Each black circle represents a different RAS circuit. (e) Best performing RAS-targeting circuits in colon cancer cells. The parts used in each RAS circuit are listed in the table below. The bar graph shows the dynamic range, while the fluorescence histograms show mCerulean-positive cells obtained in the On-(HCT-116WT) and Off-state (HCT-116k.o.) of the circuits. (f) RAS-targeting circuits are classifiers for cells with mutated RAS. Output expression of RAS-targeting circuits in different cancer cell lines with (RASMUT = blue) or without (RASWT = orange) mutation leading to increased RAS activation. The colored symbols represent biological replicates of the different cell lines. The parts used in each RAS circuit are indicated in the table below. mCerulean output expression was measured by flow cytometry and normalized to a constitutively expressed mCherry transfection control. Dynamic range was calculated as fold change between normalized output expression in HCT-116WT and HCT-116k.o.. Each circuit was measured in three biological replicates. Error bars were calculated using error propagation rules. Significance was tested using an unpaired two-tailed Student’s t-test. **p < 0.01, ***p < 0.001.