Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins

Abstract

Synthetic genetic devices that interface with native cellular pathways can be used to change natural networks to implement new forms of control and behavior. The engineering of gene networks has been limited by an inability to interface with native components. We describe a class of RNA control devices that overcome these limitations by coupling increased abundance of particular proteins to targeted gene expression events through the regulation of alternative RNA splicing. We engineered RNA devices that detect signaling through the nuclear factor κB and Wnt signaling pathways in human cells and rewire these pathways to produce new behaviors, thereby linking disease markers to noninvasive sensing and reprogrammed cellular fates. Our work provides a genetic platform that can build programmable sensing-actuation devices enabling autonomous control over cellular behavior.

Fig. 1

An alternative splicing-based RNA control device translates protein inputs to targeted gene expression outputs. (A) Platform composition of an RNA control device based on alternative splicing. The input module, consisting of an RNA-based protein sensor or aptamer, detects changes in nuclear protein concentrations. The sensor transmits information on binding events to the actuator module, consisting of a three-exon, two-intron mini-gene where the alternatively spliced exon contains a stop codon. The actuator controls the expression of the output module, consisting of a gene of interest (GOI). The three modules are physically linked in a transcript to form the assembled RNA control device. (B) Mechanism of RNA device function. A 3’ ss device is shown, where the input module is located in the intron upstream of the alternative exon and binding of the protein input to the sensor alters the splicing pattern by either enhancing (green) or suppressing (red) alternative exon inclusion. Exclusion of the alternative exon results in removal of the stop codon upstream of the GOI, thereby increasing the gene expression output from the device. (C) Determination of optimal input module location within intronic sequence space of the regulatory device. The MS2 aptamer was inserted at 12 intronic positions spaced by 15-nts flanking the alternatively spliced exon. (D) Fluorescence images of the MS2-responsive devices. The increased fluorescence output from an MS2-responsive device is specific to the MS2-DsRed protein input and the wild-type MS2 aptamer in position 3 (MS2-3). (E) The response of the MS2-responsive device to the MS2-DsRed protein is affected by the location of the input module. For all activities reported as relative expression (fold), the ratio of the mean GFP levels of the wild-type RNA device in the presence of ligand (MS2-DsRed) to the absence of ligand (DsRed) is normalized to the same ratio for the mutant device. Transcript isoform analysis of the MS2-responsive devices with qRT-PCR supported the gene expression data (bottom panel). For all qRT-PCR data reported as relative excl/incl (fold), the ratio of the mean expression levels of the exon 7 excluded isoform to the exon 7 included isoform for the wild-type device in the presence of ligand to the absence of ligand is normalized to the same ratio for the mutant device. For all reported activities, mean expression levels from two independent experiments are shown. Error bars represent +/− s.d. from mean values. P-values derived from the Student’s t-test are as follows: *P < 0.05 and **P < 0.01. Unnormalized expression levels for all devices are provided in Tables S1 to S6.

Fig. 2

RNA control devices detect endogenous protein inputs and signaling through native pathways. (A) Mechanism of the NF-κB-responsive device based on TNF-α stimulation (20 ng/ml) of the NF-κB pathway. Ligand binding to the TNF-α receptor leads to activated signaling and translocation of p50 and p65 into the nucleus. The NF-κB-responsive devices contain NF-κB p65 (p65-3) or p50 (p50(1)-3 and p50(2)-3) aptamers inserted into position 3. (B) Phase (top) and fluorescence (bottom) images of the NF-κB p65-responsive devices. The increased fluorescence output from a NF-κB-responsive device is specific to the pathway stimulation and the wild-type p65 aptamer in position 3 (p65-3). (C) NF-κB-responsive devices exhibited responses to TNF-α stimulation at the level of gene expression (left panel) and splicing pattern (right panel). For all data, relative expression (fold) was determined as described in Fig. 1F. qRT-PCR data is reported as relative excl/incl, the ratio of the mean expression levels of the exon 7 excluded isoform to the exon 7 included isoform for the wild-type device relative to the same ratio for the mutant device under the indicated ligand condition. (D) Mechanism of the NF-κB p50-responsive device based on a p50-DsRed protein input and corresponding device response. The NF-κB p50-responsive devices exhibited responses to a heterologous p50-DsRed protein similar to that observed with TNF-α stimulation. Activities were reported as relative expression by taking the ratio of the mean GFP levels of the wild-type RNA device to that from the mutant device under the indicated ligand condition. (E) Mechanism of the β-catenin-responsive device based on LTD4 stimulation (80 nM) of the Wnt pathway. LTD₄ stimulation leads to stabilization of β-catenin and accumulation in the nucleus. The β-catenin-responsive devices contain the β-catenin aptamer in positions 3 (β-cat-3) and 6 (β-cat-6). (F) β-catenin-responsive devices exhibited responses to LTD₄ stimulation at the level of gene expression (left panel) and splicing pattern (right panel).

Fig. 3

RNA devices implement combinatorial control schemes through multi-input processing. (A) Mechanism of the MS2 multi-input processing regulatory device. Wild-type and mutant MS2 aptamers were inserted into positions 3 and 10. (B) The MS2 multi-input processing device responds to the heterologous MS2-DsRed protein to increase the gene expression output (top panel). Transcript isoform analysis of the MS2 multi-input processing device supports gene expression data (bottom panel). For all data, relative expression (fold) and relative ratios of exon excluded to included transcript isoforms (fold) were determined as described in Fig. 1F. (C) The MS2 / NF-κB p50 multi-input processing regulatory device allows integration of complex input signals and amplification of device response. The NF-κB p50 and MS2 aptamers were inserted into positions 3 and 10, respectively. (D) The MS2 / NF-κB p50 multi-input processing device responds to both inputs to increase the gene expression output (top panel). Transcript isoform analysis of the MS2 / NF-κB p50 multi-input processing device supports gene expression data (bottom panel).

Fig. 4

RNA devices detect endogenous markers of disease and trigger targeted cell death. (A) Functional representation of a targeted therapeutic device that integrates across two therapeutic inputs – disease biomarker and an exogenously-applied, inactive pro-drug - to trigger targeted cell death. (B, C) Mechanisms of the β-catenin- (β-cat-6) (B) and NF-κB-responsive (p65-3) (C) devices fused to a suicide gene therapy output module (HSV-TK), which control cell survival in response to detection of disease markers and GCV, a pro-drug trigger. (D) Dose-response curves of cell survival percentages for the β-catenin- and NF-κB-responsive devices fused to HSV-TK indicate a decrease in cell survival as a result of increased signaling through the targeted pathway and the presence of GCV. For all reported data, the mean cell survival levels from two independent experiments are shown.