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Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor

  • Walter Thavarajah
    Walter Thavarajah
    Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    More by Walter Thavarajah
  • , 
  • Adam D. Silverman
    Adam D. Silverman
    Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    More by Adam D. Silverman
  • , 
  • Matthew S. Verosloff
    Matthew S. Verosloff
    Interdisciplinary Biological Sciences Graduate Program, Northwestern University, 2204 Tech Drive, Evanston, Illinois 60208, United States
    Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    More by Matthew S. Verosloff
  • , 
  • Nancy Kelley-Loughnane
    Nancy Kelley-Loughnane
    Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433, United States
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    Orcidhttp://orcid.org/0000-0003-2974-644X
  • , 
  • Michael C. Jewett
    Michael C. Jewett
    Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    More by Michael C. Jewett
    Orcidhttp://orcid.org/0000-0003-2948-6211
  • , and 
  • Julius B. Lucks*
    Julius B. Lucks
    Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    *E-mail: [email protected]
    More by Julius B. Lucks
    Orcidhttp://orcid.org/0000-0002-0619-6505
Cite this: ACS Synth. Biol. 2020, 9, 1, 10–18
Publication Date (Web):December 12, 2019
https://doi.org/10.1021/acssynbio.9b00347

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Abstract

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Advances in biosensor engineering have enabled the design of programmable molecular systems to detect a range of pathogens, nucleic acids, and chemicals. Here, we engineer and field-test a biosensor for fluoride, a major groundwater contaminant of global concern. The sensor consists of a cell-free system containing a DNA template that encodes a fluoride-responsive riboswitch regulating genes that produce a fluorescent or colorimetric output. Individual reactions can be lyophilized for long-term storage and detect fluoride at levels above 2 ppm, the Environmental Protection Agency’s most stringent regulatory standard, in both laboratory and field conditions. Through onsite detection of fluoride in a real-world water source, this work provides a critical proof-of-principle for the future engineering of riboswitches and other biosensors to address challenges for global health and the environment.

KEYWORDS:
Safe drinking water availability is an important contributor to public welfare. (1) However, safe water sources are not available to a large portion of the globe, with an estimated 3 billion people using water from either an unsafe source or a source with significant sanitary risks. (2) One particularly dangerous contaminant is fluoride, which leaches into groundwater from natural sources. Long-term exposure to fluoride concentrations above 2 ppm can cause dental and skeletal fluorosis, heavily burdening communities in resource-limited settings. (3) Though large-scale remediation strategies are available, they are resource-intensive and difficult to deploy. (3,4) This problem is compounded by the reliance of gold-standard sensing methods on expensive analytical equipment, making detection difficult in areas with the greatest need. (4) While many emerging fluorescent and colorimetric chemical fluoride sensors exist, these either require supplementary imaging equipment or utilize toxic organic solvents, hampering their use in real-world conditions. (5) To facilitate targeted remediation and empower affected individuals, there is a pressing need for a more practical, rapid, and field-deployable solution to monitor the presence of fluoride in water.
Because they can be lyophilized and rehydrated on-demand, cell-free expression (CFE) systems have recently become a promising platform for field-deployable molecular diagnostics. (6−9) These systems typically consist of cellular gene expression machinery along with the required buffers, energy sources and cofactors necessary to support gene expression from added DNA templates. (10) Unlike whole cells, cell-free platforms offer an open, easily tunable reaction environment, expediting the design process for genetically encoded programs. (10) Furthermore, they circumvent the analyte toxicity, host mutation, and biocontainment concerns limiting cellular sensors. (11)
We sought to leverage the advantages of cell-free biosensing platforms to create a new approach for monitoring for the presence of fluoride in water using a fluoride-responsive riboswitch that regulates the expression of the CrcB fluoride efflux pump in Bacillus cereus. (12) By configuring the B. cereus crcB fluoride riboswitch to control the transcription of downstream reporter genes, (13) we show that a cell-free gene expression system can activate both protein and RNA reporter expression in the presence of fluoride. With an enzymatic colorimetric reporter, we demonstrate detection of fluoride concentrations at the Environmental Protection Agency (EPA) Secondary Maximum Contaminant Level of 2 ppm. (14) Notably, these cell-free biosensors showed more accurate sensing with a lower limit of detection than several tested commercially available consumer fluoride testing kits. We also demonstrate that our fluoride biosensor can be lyophilized for long-term storage and distribution, allowing us to detect fluoride in unprocessed groundwater obtained and tested onsite in Costa Rica. This work exemplifies the potential of riboswitches as practical biosensing tools and helps lay the foundation for utilizing cell-free biosensing systems in rapid and field-deployable water quality diagnostics to address pressing challenges in global health.

Results

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Fluoride Riboswitch Control of Reporter Expression in Cell-Free Reactions

Our point-of-use diagnostic consists of a cell-free system containing a fluoride biosensor DNA template that can be lyophilized and stored. Rehydration activates the biosensor, which encodes the fluoride riboswitch and a reporter gene that produces a detectable output if fluoride is present (Figure 1a,b). As a starting point, we sought to characterize the regulatory activity of the B. cereus crcB riboswitch in the cell-free reaction environment. Previous characterization of the riboswitch’s cotranscriptional folding mechanism (Figure 1b) confirmed that it functions with E. coli RNA polymerase, (13) allowing us to use it in E. coli cell-free extract. We therefore constructed a reporter plasmid containing the riboswitch sequence followed by a strong ribosome binding site (RBS) and the coding sequence of the reporter protein superfolder green fluorescent protein (sfGFP), all placed downstream of a constitutive E. coli σ70 promoter (all plasmid details in Supplemental Table S1).

Figure 1

Figure 1. Cell-free fluoride biosensor engineering strategy. (a) Schematic for lyophilization of a cell-free reaction in tubes or on paper disks. Rehydration with a water sample allows the designed biosensing reaction to proceed to yield a detectable signal. (b) Schematic for fluoride riboswitch-mediated transcriptional regulation in cell-free extract. The riboswitch folds cotranscriptionally into one of two mutually exclusive states, depending on the presence of fluoride. In the absence of fluoride, the riboswitch folds into a terminating hairpin, precluding downstream gene expression. Fluoride binding stabilizes a pseudoknot structure (red paired region, inset from PDB: 4ENC) that sequesters the terminator and enables the expression of downstream reporter genes. (c) Schematic of a cell-free fluoride biosensor, consisting of a DNA template encoding the fluoride riboswitch controlling the expression of sfGFP. Eight-hour end point fluorescence measurements for reactions containing NaF (dark green) or NaCl (gray) are shown below. Error bars represent one standard deviation from three technical replicates.

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After optimizing the level of Mg2+ within the reaction conditions for riboswitch performance (Supplemental Figure S1), we determined the fluoride sensor’s dose–response to fluoride by titrating across a range of NaF concentrations. All tested conditions caused a measurable increase in expression over the OFF state, with activation seen at NaF concentrations as low as 0.1 mM (Figure 1c, green line and inset). This threshold is important, since 0.1 mM NaF is equivalent to the EPA’s 2 ppm secondary maximum contaminant level for fluoride in drinking water, its most stringent risk threshold. (14) However, because the signal-to-noise ratio at 0.1 mM NaF is below 3, we estimated the reliable lower limit of detection to be 0.2 mM NaF. Importantly, the system also has low leak—we observed minimal activation of gene expression in the absence of NaF. Titration of identical concentrations of NaCl showed no increase in expression at any condition, demonstrating that the riboswitch is highly specific for fluoride (Figure 1c, gray line). This result corroborates a previous and more extensive characterization in E. coli of the switch’s specificity for fluoride. (12) Thus, without any optimization of riboswitch structure or function, the sensor can discriminate health-relevant concentrations of fluoride dosed into laboratory water samples.

Changing Reporters to Tune Sensor Speed and Detection Threshold

Biosensor field deployment requires an output that can be quickly read with minimal supplemental equipment. (16) Using the maximally activating fluoride concentration (3.5 mM), reactions achieved measurable signal above the no-fluoride OFF state in 30 min at 30 °C, with overall 20-fold activation relative to the no-fluoride condition at the end of the 8-h experiment (Figure 2a). Despite this, the sensor’s ON state was not distinguishable by eye for several hours even after excitation with a blue LED, presenting the need for a faster reporter.

Figure 2

Figure 2. Riboswitch modularity allows fluorescent protein, RNA aptamer, and enzymatic colorimetric reporter outputs. Biosensor DNA template layouts and concentrations shown above reporter information and characterization data for that reporter. (a) Superfolder GFP (sfGFP) reporter (structure from PDB: 2B3P). Time course of fluorescence in the presence of 3.5 mM NaF (dark green), 0.2 mM NaF (light green), or 0 mM NaF (gray). (b) Three-way junction dimeric Broccoli reporter (structure predicted from NUPACK (15)). Time course of fluorescence in the presence of 3.5 mM NaF (dark green), 0.2 mM NaF (light green), and 0 mM NaF (gray). (c) Catechol (2,3)-dioxygenase (C23DO) reporter. Reaction scheme shows the cleavage of the colorless catechol molecule into the yellow 2-hydroxymuconate semialdehyde. Time course of absorbance at 385 nm in the presence of 3.5 mM NaF (orange), 0.2 mM NaF (yellow), and 0 mM NaF (gray). For each plot, trajectories represent average and error shading represents one standard deviation from three technical replicates. (a) and (b) are reported in mean equivalent fluorescence (MEF).

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We hypothesized that we could accelerate the sensor’s response with a 3-way junction dimeric Broccoli (3WJdB) (17) reporter, an RNA aptamer that activates fluorescence of its DFHBI-1T ligand upon transcription, eliminating delays caused by translation. At all tested NaF concentrations, 3WJdB produced a signal detectable over background within 12 min at 30 °C (Figure 2b), more than twice as fast as could be achieved with sfGFP (Figure 2a). Interestingly, this result also confirms that the fluoride riboswitch is compatible with RNA reporters, despite the potential for misfolding with the upstream riboswitch sequence. However, despite the improvement in speed, exchanging sfGFP for 3WJdB resulted in a 50-fold reduction in the sensor’s fluorescent output at the maximally activating tested condition. Thus, although the RNA-level output is preferable for its speed relative to the sfGFP output if a plate reader is accessible, it is not bright enough to use for field deployment.
As an alternative to a fluorescent output, we used the colorimetric enzyme catechol (2,3)-dioxygenase (C23DO) as a reporter. C23DO has previously been used in genetically encoded biosensors for plant viruses (18) and produces a visible reporter output by oxidizing its colorless catechol substrate to the yellow-colored 2-hydroxymuconate semialdehyde. (19) This color change allows gene expression to be read out either by light absorbance at 385 nm on a plate reader or by the appearance of a yellow color, visible to the naked eye. All tested fluoride concentrations produced a visible output within 70 min at 30 °C, which we empirically defined as an absorbance of 0.8 based on our previous observations (Figure 2c). (18) Notably, there was only a 20 min time separation between the minimally and maximally activating conditions, highlighting the ability of enzymatic reporters to quickly amplify weak signals. Consistent with previous uses of C23DO as a reporter in a cell-free reaction, (19) we observed a decay in the absorbance signal after it reached peak activation, possibly due to 2-hydroxymuconate semialdehyde degradation. This effect does not compromise sensor robustness because differences in activation for an enzymatic reporter are determined by differences in time to observable signal rather than final signal magnitude, which is determined by the amount of substrate supplied. One disadvantage of this strategy is that activation time does not linearly correlate with fluoride concentration, limiting the sensor to only supplying a binary presence/absence result within a specified time window. (16) Despite this, the sensor’s sensitivity and low leak make this presence/absence result diagnostically informative, which combined with the advantages of an easily visualized output and reasonable time to detection made C23DO our reporter of choice for a field-deployable diagnostic.

Reaction Tuning and Lyophilization toward Biosensor Field Deployment

We next took steps to optimize our sensor to detect fluoride near the EPA’s secondary maximum contaminant limit of 2 ppm (100 μM). We obtained a robust ON signal with our original design, but the sensor began to leak without fluoride after 90 min (Figure 2c, gray line), complicating detection for trace amounts of fluoride. We attempted to mitigate this problem by reducing the amount of reporter DNA supplied to the reaction from 5 nM to 3 nM to diminish the sensor’s output. In doing so, we completely suppressed leak while detecting 100 μM NaF over background (Figure 3a), but at the cost of significantly delaying activation. We could detect as low as 50 μM NaF over background in this leakless sensor, but only during an extended incubation that did not reach a visually detectable threshold within 6 h.

Figure 3

Figure 3. Colorimetric reporters enable fluoride sensing at environmentally relevant concentrations. (a) Time course of 385 nm absorbance as measured by plate reader in the presence of 100 μM NaF (orange), 50 μM NaF (yellow), and 0 μM NaF (gray) using C23DO as a reporter and incubated at 30 °C. Trajectories represent average and error shading represents one standard deviation from three technical replicates. (b) Color change observed after 1-h for two different reporter template concentrations with and without 100 μM NaF. Tubes were mixed by pipetting and incubated at 37 °C before image capture at 60 min. (c) Time lapse of rehydrated lyophilized reactions incubated at 37 °C in the absence (top) and presence (bottom) of 1 mM NaF.

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To solve this dilemma and maintain a practical incubation time, we sought a strategy whereby tests could be interpreted as “ON” only if the yellow color appeared within some externally specified time window. Under these constraints, sensor leak is not a problem as long as the difference in time scale between the ON and OFF state is suitably longer than the test time. To implement this strategy, we increased biosensor DNA concentration to 10 nM and also increased the temperature of the CFE reaction to 37 °C. Under these conditions, activation by 100 μM NaF resulted in a clear color change in 60 min with no visible leak in the OFF condition (Figure 3b, Supplemental Figure S2). The same conditions using 3 nM DNA template resulted in no color change within 60 min. This result highlights an appreciable advantage afforded by the open reaction environment of cell-free systems: the sensor’s limit of detection can be tuned simply by manipulating the reaction time and the DNA concentration of the biosensor.
Recent work demonstrates that CFE reactions can be lyophilized and rehydrated when needed for on-demand biomanufacturing, nucleic acid detection, and educational activities. (6,7,20,21) To expand these applications to point-of-use small molecule detection, we next aimed to demonstrate that fluoride biosensor reactions maintain functionality after being lyophilized. We measured the impact of lyophilization on fluoride detection by lyophilizing reactions containing 10 nM C23DO reporter plasmid overnight. The reactions were then rehydrated with laboratory grade Milli-Q water (Figure 3c, top) or water containing 1 mM NaF (Figure 3c, bottom) and incubated at 37 °C. Time-lapse photography shows visible activation within 60 min in the 1 mM NaF condition with no leak observed within 100 min in the no-fluoride condition (Supplemental Video S1). This finding, consistent with other recent reports from lyophilized cell-free systems, (6,7,20,21) indicates that sensing by the fluoride riboswitch in CFE reactions is not disrupted by the lyophilization process.
We also tested the viability of lyophilized reactions stored over longer periods of time. After lyophilization, reaction tubes were wrapped in Parafilm and stored in Drierite for 3 months in darkness at room temperature and atmospheric pressure before being removed and rehydrated with laboratory grade Milli-Q water or water containing 1 mM NaF. The sample rehydrated with 1 mM NaF showed strong activation within 1 h, with no leak observed in the no-fluoride condition (Supplemental Figure S3). Interestingly, lyophilization appeared to suppress leak in the no-fluoride condition without impacting the ability to activate expression with fluoride. The maintained viability of reactions after three months indicates that storage in desiccant and light shielding to prevent catechol oxidation are the only requirements for long-term storage of lyophilized cell-free reactions, a crucial step toward field-deployment.

Point-of-Use Detection of Environmental Fluoride with a Lyophilized Biosensor

Components of environmental water samples, particularly natural ions like sodium, magnesium, or potassium, could poison cell-free reactions upon rehydration. These “matrix effects” would then impede the translation of a sensor from lab experiments to field testing and must be accounted for in a field-deployable diagnostic. To test the robustness of our system against matrix effects, we created mock fluoride-containing field samples by sampling water from a municipal tap, Lake Michigan, and an outdoor swimming pool, with Milli-Q water used as a control. NaF was then added to each sample to a final concentration of 1 mM. The biosensing reactions were prepared as before and pipetted into PCR tubes (Figure 4a, top) or spotted on BSA-treated chromatography paper (Figure 4a, bottom) before being lyophilized overnight. After lyophilization, reactions were immediately rehydrated with either unaltered mock field sample (− condition) or mock field sample containing 1 mM NaF (+ condition) and incubated at 37 °C for 1 h. For all fluoride-containing samples both in tubes and on paper, a color change was observed within 1 h, with no color development in any of the no-fluoride conditions. These results confirm that the fluoride biosensor is robust against the unfiltered environmental samples tested and can be used in real-world conditions.

Figure 4

Figure 4. The cell-free fluoride riboswitch biosensor functions with real-world water samples and is not impacted by long-term storage and distribution. (a) Cell-free reactions rehydrated with various water samples with or without 1 mM NaF added. Lyophilized reactions in tubes are shown above lyophilized reactions on chromatography paper before and after 1 h incubation at 37 °C. MQ = laboratory grade Milli-Q water; Tap = tap water; Lake = unfiltered Lake Michigan water; Pool = unfiltered outdoor pool water. Uncropped photos of chromatography paper experiments are available in Supplemental Figure S8. (b) Field testing of lyophilized cell-free reactions rehydrated with water sampled in Cartago, Costa Rica. Geographical data © OpenStreetMap contributors. (22) The positive control contained 1 mM NaF in the reaction before lyophilization. The negative control was rehydrated with Milli-Q water, and the positive control and each test were rehydrated with 20 μL of unprocessed field sample followed by body-heat incubation for 5 h. Measured fluoride concentrations obtained using a fluoride sensing electrode. Field samples are from sites B and E in Supplemental Table S2.

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As the culmination of our optimization process, we tested our sensor’s ability to accurately classify fluoride-containing samples in the field. We specifically sought to follow a previously published environmental fluoride study that used conventional methods to sample and test publicly available natural and municipal water sources near the Irazu volcano in Cartago, Costa Rica, an area shown to have elevated fluoride levels (Supplemental Figure S4). (23) To do this, we manufactured lyophilized fluoride biosensor reactions and transported them to Costa Rica using our simplified desiccant packaging (Supplemental Figure S5A) for field testing. Sampling regions identified in the previous study, (23) we collected samples in 50 mL conical tubes and tested for fluoride in batch by adding unprocessed water to lyophilized reactions in PCR tubes via single-use exact volume transfer pipettes (Supplemental Figure S5B).
All field-testing was done onsite in Costa Rica without access to laboratory resources or equipment. Reactions were incubated at approximately 37 °C by being held in the armpit, with reaction time increased to 5 h to control for delayed activation caused by the imprecision of body heat incubation and low environmental fluoride concentrations. (18) A strong yellow color developed in every positive control reaction within an hour, confirming robustness to reaction poisoning by potential sample matrix effects (Supplemental Table S2). No activation was observed within 5 h in any samples with fluoride concentrations less than 50 μM (∼1 ppm) as measured in cross-validation with a commercial fluoride-sensing electrode. However, a visible color change was observed after 3.5 h in a water sample collected from a roadside ditch measured to have a fluoride concentration of 60 μM (Figure 4b). This delayed activation aligns with our previous characterization in detecting trace concentrations of fluoride below 100 μM (Figure 3a). For all samples, the commercial electrode measurement confirmed the conclusions drawn from the cell-free sensors, with no false positives or false negatives observed under any conditions (n = 9) (Supplemental Table S2). By accurately detecting levels of fluoride relevant to public health concern thresholds in a real-world water source with minimal supplementary equipment, we have shown that lyophilized fluoride biosensor CFE reactions can be effectively used as low-cost, point-of-use diagnostics, demonstrating the potential of engineered biosensor elements for small molecule detection in the field.

Discussion

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In this work, we have demonstrated that a fluoride riboswitch can be implemented in a CFE system to act as a field-deployable diagnostic for environmental water samples. To the best of our knowledge, this is the first demonstration of a cell-free riboswitch-based biosensor that can detect health-relevant small molecules at regulatory levels within the field. Importantly, this work represents a significant improvement in efficacy over commercially available consumer kits (Supplemental Figure S6) and provides significant simplification and cost savings over gold standard electrochemical methods of fluoride detection, which cost hundreds to thousands of dollars and are cumbersome to use even for scientifically skilled operators. In contrast, our biosensors can currently be made for $0.40/reaction, (20) only require a drop of water, and are robust to temperature variation, enabling incubation with body heat.
A key strength of cell-free biosensing is that biochemical parameters such as cofactor and DNA concentration can be easily tuned to reduce leak and improve dynamic range, which has been a historically difficult challenge for riboswitch engineering in cells. Furthermore, since riboswitches are cis-acting, only one DNA template concentration needs to be tuned per sensor, simplifying the optimization space relative to trans-acting RNA or protein regulators. When optimizing these reactions for the field, we found that reactions lyophilized in PCR tubes had advantages over paper-based reactions, which rapidly dried out even when incubated in sealed, humidified containers. This effect was exacerbated by the longer incubation times required for low analyte concentrations, variabilities in ambient temperature, and the practical difficulty of equipment-free incubation of paper sensors using body heat, making the tube format much more amenable to the challenges of field deployment.
This work also highlights the feasibility of using transcriptional riboswitch-mediated gene expression to convert weak-binding RNA aptamers into functional biosensors. We were surprised to find that the B. cereus crcB riboswitch activated so well in an E. coli cell-free lysate system, given the sophisticated nature of its folding mechanism and transcriptional readthrough observed both in vitro and in vivo (13,24) Transcriptional riboswitches often show weak activation due to the short time scales of their regulatory decision-making, resulting in sensitivities that are kinetically, rather than thermodynamically limited. (25) Coupling transcriptional riboswitches to enzymatic outputs like C23DO can amplify weak signals, since each reporter enzyme turns over multiple molecules of substrate. (26) The combined kinetic mechanism of switching and the signal amplification afforded by a colorimetric reporter resulted in our sensor achieving a limit of detection of 50 μM, less than half of the lowest previously measured KD for any fluoride aptamer. (27) Thus, this work is a powerful example of why considering only thermodynamic binding affinities during aptamer selection can exclude promising, diagnostically relevant sensors.
The strategies we present here could be applied to optimize the performance of a large number of natural riboswitches for the detection of metabolites and ions relevant to environmental and human health monitoring. (28) Additionally, the compatibility of CFE reactions for high-throughput screening (29) and the simple format of our DNA expression construct could be used to characterize the thousands of “orphan” riboswitches that have been bioinformatically identified but bind to unknown ligands. (30) We imagine that these strategies could even be used to re-engineer riboswitches to have novel function. (31−33) As the rules of riboswitch mechanisms are deciphered at deeper levels, (13,34−36) we hope to reach a sufficient understanding to design their functional properties to meet the global needs for field-deployable environmental and health diagnostics.

Materials and Methods

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Plasmid Construction

Plasmids were assembled using Gibson assembly (New England Biolabs, Cat#E2611S) and purified using a Qiagen QIAfilter Midiprep Kit (QIAGEN, Cat#12143). pJBL7025 and pJBL7026 were assembled from pJBL3752. A table of all plasmid sequences can be found in Supplemental Table S1.

Extract Preparation

Extracts were prepared according to published protocols using sonication and postlysis processing in the Rosetta2 (DE3) pLysS strain. (10) Briefly, cells are plated on a chloramphenicol-selective agar plate and incubated overnight then used to inoculate a 20 mL overnight starter culture for a 1 L final culture. This culture is grown to an optical density (OD600) of 3.0 ± 0.2 then pelleted and lysed by sonication before centrifugation for 10 min at 4 °C and 12 000g. After lysis, extracts were incubated with shaking for 80 min at 37 °C and 200 rpm then recentrifuged under the same conditions. The supernatant was injected into a 10K MWCO dialysis cassette (Thermo Fisher, 66380) and dialyzed at 4 °C for 3 h before a final centrifugation under the same conditions and snap-freezing in liquid nitrogen.

CFE Experiment

CFE reactions were prepared according to established protocols. (10) Briefly, reactions are composed of cell extract, a reaction buffer containing NTPs, amino acids, buffering salts, crowding agents, and an energy source, and a mix of template DNA and inducers in an approximately 30/30/40 ratio. Between reactions, the only conditions varied are DNA template and concentration, inducer concentration, and buffering magnesium glutamate concentration, the last of which is optimized by extract. Optimal magnesium glutamate concentration was 20 mM for shelf stability and field deployment experiments and 12 mM for all other data. Little variability was seen in extract performance between batches using the appropriate optimal magnesium concentrations (Supplemental Figure S7).
For an example reaction setup, refer to the Supplemental Experimental Design Spreadsheet. All kinetic CFE reactions were prepared on ice in triplicate at the 10 μL scale. 33 μL of a mixture containing the desired reaction components was prepared and then 10 μL was pipetted into three wells of a 384-well plate (Corning, 3712), taking care to avoid bubbles. Plates were sealed (Thermo Scientific, 232701) and kinetic data was monitored on a BioTek Synergy H1m plate reader for sfGFP (20 nM reporter plasmid, emission/excitation: 485/520 nm every 5 min for 8 h at 30 °C), C23DO (variable reporter plasmid concentration, 385 nm absorbance every 30 s for 4–6 h at 30 °C), and 3WJdB (20 nM reporter plasmid, emission/excitation 472/507 nm every 30 s for 2 h at 30 °C). C23DO reactions were supplemented with 1 mM catechol and 3WJdB reactions were supplemented with 20 μM DFHBI-1T. For all fluorescence experiments, a no-DNA negative control was prepared in triplicate for every extract being tested. All reported fluorescence values have been baseline-subtracted by the average of three samples from the no-DNA condition. Baseline subtraction was not performed for catechol reactions because reaction progress is determined from time to activation rather than maximal absorbance value. For the data depicted in Figure 1c, NaF and NaCl titrations were performed in separate experiments.

Mean Equivalent Fluorescence Calibration

Fluorescence measurements were calibrated to a standard curve of fluorescein isothiocyanate (FITC) fluorescence to give standardized fluorescence units of μM equivalent FITC following a previously established procedure. (37) Briefly, serial dilutions were performed from a 50 μM stock and prepared in a pH 9.5, 100 mM sodium borate buffer. Fluorescence values for these samples were read at an excitation wavelength of 485 nm and emission wavelength of 520 nm for sfGFP and an excitation wavelength of 472 nm and emission wavelength of 507 nm for 3WJdB. These values were then used to calculate a linear conversion factor relating the plate reader’s output in arbitrary units to the FITC standard curve.

Lyophilization

All lyophilization was performed in a Labconco FreeZone 2.5 Liter −84 °C Benchtop Freeze-Dryer (Cat# 710201000). A CFE reaction master mix was prepared and split into 20 μL aliquots in PCR strip tubes. Tube caps were then pierced with a pin and strips were wrapped in aluminum foil before being flash frozen in liquid nitrogen and lyophilized overnight at 0.04 mbar. After lyophilization, pierced PCR strip tube caps were replaced. Tubes were then sealed with parafilm and placed directly into Drierite (Cat#11001) for storage at room temperature (Supplemental Figure S5A).

Paper Sensors

Individual sensors were punched out of Whatman 1 CHR chromatography paper (3001–861) using a Swingline Commercial Desktop Punch (A7074020). Tickets were then placed in a Petri dish and immersed in 4% BSA for 1 h before being transferred to a new dish and left to air-dry overnight. After drying, tickets were spotted with 20 μL of CFE reaction and placed in plastic jars (QOSMEDIX 29258), which were loosely capped and wrapped in aluminum foil before being flash frozen in liquid nitrogen and lyophilized overnight at 0.04 mbar. For testing, tickets were transferred to new jars and rehydrated with 20 μL of sample solution. Jars were then closed and sealed with parafilm before incubation for 1 h at 37 °C.

Field Deployment

20 μL lyophilized reactions were prepared with 10 nM pJBL7025 and 1 mM catechol. As a positive control, additional reactions were lyophilized after being pre-enriched with 1 mM NaF. Supplemental Table S2 contains a complete list of sample site locations and water sources tested. 50 mL water samples were collected and stored in Falcon tubes (Fisher Scientific, Cat# 14-432-22) without any processing or filtration. Reactions were rehydrated by using 20 μL exact volume transfer pipettes (Thomas Scientific, 1207F80) to pull from collected samples. Three reactions were run at each sample site: (1) a positive control rehydrated with the sample, (2) a blank reaction rehydrated with the sample, and (3) a negative control reaction rehydrated with purified water to test for any reaction leak. Reactions were placed in a plastic bag and incubated at body temperature in the armpit for 5 h using established protocols and marked as activated if a visible yellow color was observed. (18) Quantitative measurements of fluoride concentration of the same sample were taken with an Extech ExStik Waterproof Fluoride Meter (Cat# FL700). Geographical data from OpenStreetMap is licensed under the Open Data Commons Open Database License (https://www.openstreetmap.org/copyright).

Image Capture

All images were captured with via cell phone camera, with no specialized photography setup and no postcapture editing done aside from cropping image borders. Tubes were illuminated from below via desk lamp to highlight reaction color change. Paper sensors were illuminated from above via desk lamp and photographed without removal from the plastic jars used for incubation.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.9b00347.

  • Supplemental Figures S1–S8, Supplemental Tables S1 and S2 (PDF)

  • Supplemental experimental design spreadsheet (XLSX)

  • Supplemental Video S1: Time lapse of sensor activation depicted in Figure 3c; Tubes were rehydrated with either water (left) or 1 mM NaF (right); Total time elapsed is 100 minutes (MP4)

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Author Information

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  • Corresponding Author
    • Julius B. Lucks - Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Orcidhttp://orcid.org/0000-0002-0619-6505;  Email: [email protected]
  • Authors
    • Walter Thavarajah - Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    • Adam D. Silverman - Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    • Matthew S. Verosloff - Interdisciplinary Biological Sciences Graduate Program, Northwestern University, 2204 Tech Drive, Evanston, Illinois 60208, United States;  Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Water Research, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
    • Nancy Kelley-Loughnane - Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433, United States;  Orcidhttp://orcid.org/0000-0003-2974-644X
    • Michael C. Jewett - Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Center for Synthetic Biology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States;  Orcidhttp://orcid.org/0000-0003-2948-6211
  • Notes
    The authors declare the following competing financial interest(s): The authors have submitted one provisional patent application (U.S. Patent Application Serial No. 62/813,368) for the technologically important developments included in this work. J.B.L is a cofounder of Stemloop, Inc. J.B.L.s interests are reviewed and managed by Northwestern University in accordance with their conflict of interest policies.

    All source data for the main and SI figures were deposited open access in Northwestern’s Arch database (https://arch.library.northwestern.edu). Data can be accessed via DOI: 10.21985/N2RJ64.


    All plasmids are deposited in Addgene with accession numbers 128809–128811.

Acknowledgments

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We would like to thank Professor Ana Gabriela Calderón Cornejo (Universidad de Costa Rica) and Eduardo Quirós Morales for assistance with biosensor field-testing. We also thank Ashty Karim (Northwestern University) and Professor Robert Batey (University of Colorado, Boulder) for helpful comments in preparing the manuscript, along with Khalid Alam (Stemloop, Inc.) for editing the supplemental video, Jaeyoung Jung (Northwestern University) for assistance with designing the graphical abstract, and Professor Thomas Shahady (University of Lynchburg) for helpful comments about water sampling in Costa Rica. This work was supported by the Air Force Research Laboratory Center of Excellence for Advanced Bioprogrammable Nanomaterials (C-ABN) Grant FA8650-15-2-5518 (to M.C.J. and J.B.L), the David and Lucile Packard Foundation (to M.C.J.), an NSF CAREER Award (1452441 to J.B.L.), and the Camille Dreyfus Teacher-Scholar Program (to M.C.J. and J.B.L.). A.D.S. was supported in part by the National Institutes of Health Training Grant (T32GM008449) through Northwestern University’s Biotechnology Training Program. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the Air Force Research Laboratory, Air Force Office of Scientific Research, or US Government.

References

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    Computational design of small transcription activating RNAs for versatile and dynamic gene regulation
    Chappell James; Lucks Julius B; Westbrook Alexandra; Verosloff Matthew; Lucks Julius B
    Nature communications (2017), 8 (1), 1051 ISSN:.
    A longstanding goal of synthetic biology has been the programmable control of cellular functions. Central to this is the creation of versatile regulatory toolsets that allow for programmable control of gene expression. Of the many regulatory molecules available, RNA regulators offer the intriguing possibility of de novo design-allowing for the bottom-up molecular-level design of genetic control systems. Here we present a computational design approach for the creation of a bacterial regulator called Small Transcription Activating RNAs (STARs) and create a library of high-performing and orthogonal STARs that achieve up to ~ 9000-fold gene activation. We demonstrate the versatility of these STARs-from acting synergistically with existing constitutive and inducible regulators, to reprogramming cellular phenotypes and controlling multigene metabolic pathway expression. Finally, we combine these new STARs with themselves and CRISPRi transcriptional repressors to deliver new types of RNA-based genetic circuitry that allow for sophisticated and temporal control of gene expression.
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    Zhao, Bo; Guffy, Sharon L.; Williams, Benfeard; Zhang, Qi
    Nature Chemical Biology (2017), 13 (9), 968-974CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in soln. with and without ligand. Using chem.-exchange satn. transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (∼1%) and short-lived (∼3 ms) excited conformational state that unravels a conserved 'linchpin' base pair to signal transcription termination. Upon fluoride binding, this highly localized, fleeting process is allosterically suppressed, which activates transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity responses across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation.
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    Wickiser, J. Kenneth; Winkler, Wade C.; Breaker, Ronald R.; Crothers, Donald M.
    Molecular Cell (2005), 18 (1), 49-60CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
    Riboswitches are genetic control elements that usually reside in untranslated regions of mRNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A FMN (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodn. equil. with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "mol. fuse.". This reliance on the kinetics of ligand assocn. and RNA polymn. speed might be common for riboswitches that utilize transcription termination mechanisms.
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    Physical Review Letters (2011), 106 (4), 048104/1-048104/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
    A complete gene expression reaction is reconstituted in a cell-free system comprising the entire endogenous transcription, translation, as well as mRNA and protein degrdn. machinery of E. coli. In dissecting the major reaction steps, we derive a coarse-grained enzymic description of biosynthesis and degrdn., from which ten relevant rate consts. and concns. are detd. Governed by zeroth-order degrdn., protein expression follows a sharp transition from undetectable levels to const.-rate accumulation, without reaching steady state.
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    Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch
    Ren, Aiming; Rajashankar, Kanagalaghatta R.; Patel, Dinshaw J.
    Nature (London, United Kingdom) (2012), 486 (7401), 85-89CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A·U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg2+ ions, which in turn are octahedrally coordinated to water mols. and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism.
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    McCown, Phillip J.; Corbino, Keith A.; Stav, Shira; Sherlock, Madeline E.; Breaker, Ronald R.
    RNA (2017), 23 (7), 995-1011CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
    Riboswitches are commonly used by bacteria to detect a variety of metabolites and ions to regulate gene expression. To date, nearly 40 different classes of riboswitches have been discovered, exptl. validated, and modeled at at. resoln. in complex with their cognate ligands. The research findings produced since the first riboswitch validation reports in 2002 reveal that these noncoding RNA domains exploit many different structural features to create binding pockets that are extremely selective for their target ligands. Some riboswitch classes are very common and are present in bacteria from nearly all lineages, whereas others are exceedingly rare and appear in only a few species whose DNA has been sequenced. Presented herein are the consensus sequences, structural models, and phylogenetic distributions for all validated riboswitch classes. Based on our findings, we predict that there are potentially many thousands of distinct bacterial riboswitch classes remaining to be discovered, but that the rarity of individual undiscovered classes will make it increasingly difficult to find addnl. examples of this RNA-based sensory and gene control mechanism.
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    Moore, S. J., MacDonald, J. T., Wienecke, S., Ishwarbhai, A., Tsipa, A., Aw, R., Kylilis, N., Bell, D. J., McClymont, D. W., Jensen, K. (2018) Rapid Acquisition and Model-Based Analysis of Cell-Free Transcription–Translation Reactions from Nonmodel Bacteria. Proc. Natl. Acad. Sci. U. S. A. 115 (19), E4340,  DOI: 10.1073/pnas.1715806115
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    Moore, Simon J.; MacDonald, James T.; Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Polizzi, Karen M.; Biedendieck, Rebekka; Freemont, Paul S.
    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (19), E4340-E4349CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-pos. bacterium with potential biotechnol. applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quant. models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple exptl. conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition expt. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based anal. of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biol. and biotechnol. applications.
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    Greenlee, E. B., Stav, S., Atilho, R. M., Brewer, K. I., Harris, K. A., Malkowski, S. N., Mirihana Arachchilage, G., Perkins, K. R., Sherlock, M. E., and Breaker, R. R. (2018) Challenges of Ligand Identification for the Second Wave of Orphan Riboswitch Candidates. RNA Biol. 15 (3), 377– 390,  DOI: 10.1080/15476286.2017.1403002
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    Greenlee Etienne B; Stav Shira; Harris Kimberly A; Perkins Kevin R; Breaker Ronald R; Atilho Ruben M; Brewer Kenneth I; Sherlock Madeline E; Breaker Ronald R; Malkowski Sarah N; Mirihana Arachchilage Gayan; Breaker Ronald R
    RNA biology (2018), 15 (3), 377-390 ISSN:.
    Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn(2+), and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.
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    Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch
    Boussebayle, Adrien; Torka, Daniel; Ollivaud, Sandra; Braun, Johannes; Bofill-Bosch, Cristina; Dombrowski, Max; Groher, Florian; Hamacher, Kay; Suess, Beatrix
    Nucleic Acids Research (2019), 47 (9), 4883-4895CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    The development of synthetic riboswitches has always been a challenge. Although a no. of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small mol.-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related mols. both in vitro and in vivo. A detailed structure-function anal. confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.
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    Espah Borujeni, A., Mishler, D. M., Wang, J., Huso, W., and Salis, H. M. (2016) Automated Physics-Based Design of Synthetic Riboswitches from Diverse RNA Aptamers. Nucleic Acids Res. 44 (1), 1– 13,  DOI: 10.1093/nar/gkv1289
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    Automated physics-based design of synthetic riboswitches from diverse RNA aptamers
    Espah Borujeni Amin; Huso Walker; Mishler Dennis M; Wang Jingzhi; Salis Howard M
    Nucleic acids research (2016), 44 (1), 1-13 ISSN:.
    Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications.
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    Wu, M. J., Andreasson, J. O. L., Kladwang, W., Greenleaf, W. J., and Das, R. (2019) Automated Design of Diverse Stand-Alone Riboswitches. ACS Synth. Biol. 8 (8), 1838– 1846,  DOI: 10.1021/acssynbio.9b00142
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    Automated design of diverse stand-alone riboswitches
    Wu, Michelle J.; Andreasson, Johan O. L.; Kladwang, Wipapat; Greenleaf, William; Das, Rhiju
    ACS Synthetic Biology (2019), 8 (8), 1838-1846CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    Riboswitches that couple binding of ligands to conformational changes offer sensors and control elements for RNA synthetic biol. and medical biotechnol. However, design of these riboswitches has required expert intuition or software specialized to transcription or translation outputs; design has been particularly challenging for applications in which the riboswitch output cannot be amplified by other mol. machinery. We present a fully automated design method called RiboLogic for such "stand-alone" riboswitches and test it via high-throughput expts. on 2875 mols. using RNA-MaP (RNA on a massively parallel array) technol. These mols. consistently modulate their affinity to the MS2 bacteriophage coat protein upon binding of FMN, tryptophan, theophylline, and microRNA miR-208a, achieving activation ratios of up to 20 and significantly better performance than control designs. By encompassing a wide diversity of stand-alone switches and highly quant. data, the resulting ribol.-solves exptl. data set provides a rich resource for further improvement of riboswitch models and design methods.
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    Direct Observation of Cotranscriptional Folding in an Adenine Riboswitch
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    Science (Washington, DC, United States) (2012), 338 (6105), 397-400CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Growing RNA chains fold cotranscriptionally as they are synthesized by RNA polymerase. Riboswitches, which regulate gene expression by adopting alternative RNA folds, are sensitive to cotranscriptional events. We developed an optical-trapping assay to follow the cotranscriptional folding of a nascent RNA and used it to monitor individual transcripts of the pbuE adenine riboswitch, visualizing distinct folding transitions. We report a particular folding signature for the riboswitch aptamer whose presence directs the gene-regulatory transcription outcome, and we measured the termination frequency as a function of adenine level and tension applied to the RNA. Our results demonstrate that the outcome is kinetically controlled. These expts. furnish a means to observe conformational switching in real time and enable the precise mapping of events during cotranscriptional folding.
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    Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways, and the mechanisms that enable transit between them, remain largely unknown. Here, we det. the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA mols. navigate cotranscriptional folding pathways.
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  • Abstract

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    Figure 1

    Figure 1. Cell-free fluoride biosensor engineering strategy. (a) Schematic for lyophilization of a cell-free reaction in tubes or on paper disks. Rehydration with a water sample allows the designed biosensing reaction to proceed to yield a detectable signal. (b) Schematic for fluoride riboswitch-mediated transcriptional regulation in cell-free extract. The riboswitch folds cotranscriptionally into one of two mutually exclusive states, depending on the presence of fluoride. In the absence of fluoride, the riboswitch folds into a terminating hairpin, precluding downstream gene expression. Fluoride binding stabilizes a pseudoknot structure (red paired region, inset from PDB: 4ENC) that sequesters the terminator and enables the expression of downstream reporter genes. (c) Schematic of a cell-free fluoride biosensor, consisting of a DNA template encoding the fluoride riboswitch controlling the expression of sfGFP. Eight-hour end point fluorescence measurements for reactions containing NaF (dark green) or NaCl (gray) are shown below. Error bars represent one standard deviation from three technical replicates.

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    Figure 2

    Figure 2. Riboswitch modularity allows fluorescent protein, RNA aptamer, and enzymatic colorimetric reporter outputs. Biosensor DNA template layouts and concentrations shown above reporter information and characterization data for that reporter. (a) Superfolder GFP (sfGFP) reporter (structure from PDB: 2B3P). Time course of fluorescence in the presence of 3.5 mM NaF (dark green), 0.2 mM NaF (light green), or 0 mM NaF (gray). (b) Three-way junction dimeric Broccoli reporter (structure predicted from NUPACK (15)). Time course of fluorescence in the presence of 3.5 mM NaF (dark green), 0.2 mM NaF (light green), and 0 mM NaF (gray). (c) Catechol (2,3)-dioxygenase (C23DO) reporter. Reaction scheme shows the cleavage of the colorless catechol molecule into the yellow 2-hydroxymuconate semialdehyde. Time course of absorbance at 385 nm in the presence of 3.5 mM NaF (orange), 0.2 mM NaF (yellow), and 0 mM NaF (gray). For each plot, trajectories represent average and error shading represents one standard deviation from three technical replicates. (a) and (b) are reported in mean equivalent fluorescence (MEF).

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    Figure 3

    Figure 3. Colorimetric reporters enable fluoride sensing at environmentally relevant concentrations. (a) Time course of 385 nm absorbance as measured by plate reader in the presence of 100 μM NaF (orange), 50 μM NaF (yellow), and 0 μM NaF (gray) using C23DO as a reporter and incubated at 30 °C. Trajectories represent average and error shading represents one standard deviation from three technical replicates. (b) Color change observed after 1-h for two different reporter template concentrations with and without 100 μM NaF. Tubes were mixed by pipetting and incubated at 37 °C before image capture at 60 min. (c) Time lapse of rehydrated lyophilized reactions incubated at 37 °C in the absence (top) and presence (bottom) of 1 mM NaF.

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    Figure 4

    Figure 4. The cell-free fluoride riboswitch biosensor functions with real-world water samples and is not impacted by long-term storage and distribution. (a) Cell-free reactions rehydrated with various water samples with or without 1 mM NaF added. Lyophilized reactions in tubes are shown above lyophilized reactions on chromatography paper before and after 1 h incubation at 37 °C. MQ = laboratory grade Milli-Q water; Tap = tap water; Lake = unfiltered Lake Michigan water; Pool = unfiltered outdoor pool water. Uncropped photos of chromatography paper experiments are available in Supplemental Figure S8. (b) Field testing of lyophilized cell-free reactions rehydrated with water sampled in Cartago, Costa Rica. Geographical data © OpenStreetMap contributors. (22) The positive control contained 1 mM NaF in the reaction before lyophilization. The negative control was rehydrated with Milli-Q water, and the positive control and each test were rehydrated with 20 μL of unprocessed field sample followed by body-heat incubation for 5 h. Measured fluoride concentrations obtained using a fluoride sensing electrode. Field samples are from sites B and E in Supplemental Table S2.

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      PLANT-Dx: A Molecular Diagnostic for Point-of-Use Detection of Plant Pathogens
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      Rojas Zuniga, F., Floor, G., Malavassi, E., Martinez Cruz, M., and Van Bergen, M. (2014) Fluorosis Dental En La Población Infantil En Las Cercanías Del Volcán Irazú, Costa Rica. Congr. Latinoam. Estud. Química Paraguay.
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      Zhao, B., Guffy, S. L., Williams, B., and Zhang, Q. (2017) An Excited State Underlies Gene Regulation of a Transcriptional Riboswitch. Nat. Chem. Biol. 13 (9), 968– 974,  DOI: 10.1038/nchembio.2427
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      An excited state underlies gene regulation of a transcriptional riboswitch
      Zhao, Bo; Guffy, Sharon L.; Williams, Benfeard; Zhang, Qi
      Nature Chemical Biology (2017), 13 (9), 968-974CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in soln. with and without ligand. Using chem.-exchange satn. transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (∼1%) and short-lived (∼3 ms) excited conformational state that unravels a conserved 'linchpin' base pair to signal transcription termination. Upon fluoride binding, this highly localized, fleeting process is allosterically suppressed, which activates transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity responses across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation.
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      Wickiser, J. K., Winkler, W. C., Breaker, R. R., and Crothers, D. M. (2005) The Speed of RNA Transcription and Metabolite Binding Kinetics Operate an FMN Riboswitch. Mol. Cell 18 (1), 49– 60,  DOI: 10.1016/j.molcel.2005.02.032
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      Wickiser, J. Kenneth; Winkler, Wade C.; Breaker, Ronald R.; Crothers, Donald M.
      Molecular Cell (2005), 18 (1), 49-60CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)
      Riboswitches are genetic control elements that usually reside in untranslated regions of mRNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A FMN (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodn. equil. with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "mol. fuse.". This reliance on the kinetics of ligand assocn. and RNA polymn. speed might be common for riboswitches that utilize transcription termination mechanisms.
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      Karzbrun, Eyal; Shin, Jonghyeon; Bar-Ziv, Roy H.; Noireaux, Vincent
      Physical Review Letters (2011), 106 (4), 048104/1-048104/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      A complete gene expression reaction is reconstituted in a cell-free system comprising the entire endogenous transcription, translation, as well as mRNA and protein degrdn. machinery of E. coli. In dissecting the major reaction steps, we derive a coarse-grained enzymic description of biosynthesis and degrdn., from which ten relevant rate consts. and concns. are detd. Governed by zeroth-order degrdn., protein expression follows a sharp transition from undetectable levels to const.-rate accumulation, without reaching steady state.
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      Ren, A., Rajashankar, K. R., and Patel, D. J. (2012) Fluoride Ion Encapsulation by Mg 2+ Ions and Phosphates in a Fluoride Riboswitch. Nature 486 (7401), 85,  DOI: 10.1038/nature11152
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      Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch
      Ren, Aiming; Rajashankar, Kanagalaghatta R.; Patel, Dinshaw J.
      Nature (London, United Kingdom) (2012), 486 (7401), 85-89CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Significant advances in our understanding of RNA architecture, folding and recognition have emerged from structure-function studies on riboswitches, non-coding RNAs whose sensing domains bind small ligands and whose adjacent expression platforms contain RNA elements involved in the control of gene regulation. We now report on the ligand-bound structure of the Thermotoga petrophila fluoride riboswitch, which adopts a higher-order RNA architecture stabilized by pseudoknot and long-range reversed Watson-Crick and Hoogsteen A·U pair formation. The bound fluoride ion is encapsulated within the junctional architecture, anchored in place through direct coordination to three Mg2+ ions, which in turn are octahedrally coordinated to water mols. and five inwardly pointing backbone phosphates. Our structure of the fluoride riboswitch in the bound state shows how RNA can form a binding pocket selective for fluoride, while discriminating against larger halide ions. The T. petrophila fluoride riboswitch probably functions in gene regulation through a transcription termination mechanism.
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      McCown, P. J., Corbino, K. A., Stav, S., Sherlock, M. E., and Breaker, R. R. (2017) Riboswitch Diversity and Distribution. RNA 23 (7), 995– 1011,  DOI: 10.1261/rna.061234.117
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      Riboswitch diversity and distribution
      McCown, Phillip J.; Corbino, Keith A.; Stav, Shira; Sherlock, Madeline E.; Breaker, Ronald R.
      RNA (2017), 23 (7), 995-1011CODEN: RNARFU; ISSN:1355-8382. (Cold Spring Harbor Laboratory Press)
      Riboswitches are commonly used by bacteria to detect a variety of metabolites and ions to regulate gene expression. To date, nearly 40 different classes of riboswitches have been discovered, exptl. validated, and modeled at at. resoln. in complex with their cognate ligands. The research findings produced since the first riboswitch validation reports in 2002 reveal that these noncoding RNA domains exploit many different structural features to create binding pockets that are extremely selective for their target ligands. Some riboswitch classes are very common and are present in bacteria from nearly all lineages, whereas others are exceedingly rare and appear in only a few species whose DNA has been sequenced. Presented herein are the consensus sequences, structural models, and phylogenetic distributions for all validated riboswitch classes. Based on our findings, we predict that there are potentially many thousands of distinct bacterial riboswitch classes remaining to be discovered, but that the rarity of individual undiscovered classes will make it increasingly difficult to find addnl. examples of this RNA-based sensory and gene control mechanism.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2gt73M&md5=0348b51fab63f412f9ef41b449ccdf68
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      Moore, S. J., MacDonald, J. T., Wienecke, S., Ishwarbhai, A., Tsipa, A., Aw, R., Kylilis, N., Bell, D. J., McClymont, D. W., Jensen, K. (2018) Rapid Acquisition and Model-Based Analysis of Cell-Free Transcription–Translation Reactions from Nonmodel Bacteria. Proc. Natl. Acad. Sci. U. S. A. 115 (19), E4340,  DOI: 10.1073/pnas.1715806115
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      Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria
      Moore, Simon J.; MacDonald, James T.; Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Polizzi, Karen M.; Biedendieck, Rebekka; Freemont, Paul S.
      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (19), E4340-E4349CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-pos. bacterium with potential biotechnol. applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quant. models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple exptl. conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition expt. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based anal. of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biol. and biotechnol. applications.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvVGhtLjE&md5=dbedcea611b3a74066fd149ce658ada6
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      Greenlee, E. B., Stav, S., Atilho, R. M., Brewer, K. I., Harris, K. A., Malkowski, S. N., Mirihana Arachchilage, G., Perkins, K. R., Sherlock, M. E., and Breaker, R. R. (2018) Challenges of Ligand Identification for the Second Wave of Orphan Riboswitch Candidates. RNA Biol. 15 (3), 377– 390,  DOI: 10.1080/15476286.2017.1403002
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      Greenlee Etienne B; Stav Shira; Harris Kimberly A; Perkins Kevin R; Breaker Ronald R; Atilho Ruben M; Brewer Kenneth I; Sherlock Madeline E; Breaker Ronald R; Malkowski Sarah N; Mirihana Arachchilage Gayan; Breaker Ronald R
      RNA biology (2018), 15 (3), 377-390 ISSN:.
      Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn(2+), and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.
      >> More from SciFinder ®
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      Boussebayle, A., Torka, D., Ollivaud, S., Braun, J., Bofill-Bosch, C., Dombrowski, M., Groher, F., Hamacher, K., and Suess, B. (2019) Next-Level Riboswitch Development—Implementation of Capture-SELEX Facilitates Identification of a New Synthetic Riboswitch. Nucleic Acids Res. 47 (9), 4883– 4895,  DOI: 10.1093/nar/gkz216
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      Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch
      Boussebayle, Adrien; Torka, Daniel; Ollivaud, Sandra; Braun, Johannes; Bofill-Bosch, Cristina; Dombrowski, Max; Groher, Florian; Hamacher, Kay; Suess, Beatrix
      Nucleic Acids Research (2019), 47 (9), 4883-4895CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      The development of synthetic riboswitches has always been a challenge. Although a no. of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small mol.-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related mols. both in vitro and in vivo. A detailed structure-function anal. confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.
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      Espah Borujeni, A., Mishler, D. M., Wang, J., Huso, W., and Salis, H. M. (2016) Automated Physics-Based Design of Synthetic Riboswitches from Diverse RNA Aptamers. Nucleic Acids Res. 44 (1), 1– 13,  DOI: 10.1093/nar/gkv1289
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      Automated physics-based design of synthetic riboswitches from diverse RNA aptamers
      Espah Borujeni Amin; Huso Walker; Mishler Dennis M; Wang Jingzhi; Salis Howard M
      Nucleic acids research (2016), 44 (1), 1-13 ISSN:.
      Riboswitches are shape-changing regulatory RNAs that bind chemicals and regulate gene expression, directly coupling sensing to cellular actuation. However, it remains unclear how their sequence controls the physics of riboswitch switching and activation, particularly when changing the ligand-binding aptamer domain. We report the development of a statistical thermodynamic model that predicts the sequence-structure-function relationship for translation-regulating riboswitches that activate gene expression, characterized inside cells and within cell-free transcription-translation assays. Using the model, we carried out automated computational design of 62 synthetic riboswitches that used six different RNA aptamers to sense diverse chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and activated gene expression by up to 383-fold. The model explains how aptamer structure, ligand affinity, switching free energy and macromolecular crowding collectively control riboswitch activation. Our model-based approach for engineering riboswitches quantitatively confirms several physical mechanisms governing ligand-induced RNA shape-change and enables the development of cell-free and bacterial sensors for diverse applications.
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      Wu, M. J., Andreasson, J. O. L., Kladwang, W., Greenleaf, W. J., and Das, R. (2019) Automated Design of Diverse Stand-Alone Riboswitches. ACS Synth. Biol. 8 (8), 1838– 1846,  DOI: 10.1021/acssynbio.9b00142
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      Automated design of diverse stand-alone riboswitches
      Wu, Michelle J.; Andreasson, Johan O. L.; Kladwang, Wipapat; Greenleaf, William; Das, Rhiju
      ACS Synthetic Biology (2019), 8 (8), 1838-1846CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      Riboswitches that couple binding of ligands to conformational changes offer sensors and control elements for RNA synthetic biol. and medical biotechnol. However, design of these riboswitches has required expert intuition or software specialized to transcription or translation outputs; design has been particularly challenging for applications in which the riboswitch output cannot be amplified by other mol. machinery. We present a fully automated design method called RiboLogic for such "stand-alone" riboswitches and test it via high-throughput expts. on 2875 mols. using RNA-MaP (RNA on a massively parallel array) technol. These mols. consistently modulate their affinity to the MS2 bacteriophage coat protein upon binding of FMN, tryptophan, theophylline, and microRNA miR-208a, achieving activation ratios of up to 20 and significantly better performance than control designs. By encompassing a wide diversity of stand-alone switches and highly quant. data, the resulting ribol.-solves exptl. data set provides a rich resource for further improvement of riboswitch models and design methods.
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      Frieda, K. L. and Block, S. M. (2012) Direct Observation of Cotranscriptional Folding in an Adenine Riboswitch. Science (Washington, DC, U. S.) 338 (6105), 397– 400,  DOI: 10.1126/science.1225722
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      Direct Observation of Cotranscriptional Folding in an Adenine Riboswitch
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      Science (Washington, DC, United States) (2012), 338 (6105), 397-400CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Growing RNA chains fold cotranscriptionally as they are synthesized by RNA polymerase. Riboswitches, which regulate gene expression by adopting alternative RNA folds, are sensitive to cotranscriptional events. We developed an optical-trapping assay to follow the cotranscriptional folding of a nascent RNA and used it to monitor individual transcripts of the pbuE adenine riboswitch, visualizing distinct folding transitions. We report a particular folding signature for the riboswitch aptamer whose presence directs the gene-regulatory transcription outcome, and we measured the termination frequency as a function of adenine level and tension applied to the RNA. Our results demonstrate that the outcome is kinetically controlled. These expts. furnish a means to observe conformational switching in real time and enable the precise mapping of events during cotranscriptional folding.
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      Drogalis, L. K. and Batey, R. T. (2018) Requirements for Efficient Cotranscriptional Regulatory Switching in Designed Variants of the Bacillus subtilis PbuE Adenine-Responsive Riboswitch. bioRxiv  DOI: 10.1101/372573
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      Nature Chemical Biology (2019), 15 (11), 1067-1076CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
      Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways, and the mechanisms that enable transit between them, remain largely unknown. Here, we det. the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA mols. navigate cotranscriptional folding pathways.
      >> More from SciFinder ®
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      Alam, K. K., Jung, J. K., Verosloff, M. S., Clauer, P. R., Lee, J. W., Capdevila, D. A., Pastén, P. A., Giedroc, D. P., Collins, J. J., and Lucks, J. B. (2019) Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription. bioRxiv  DOI: 10.1101/619296
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    • Supplemental Video S1: Time lapse of sensor activation depicted in Figure 3c; Tubes were rehydrated with either water (left) or 1 mM NaF (right); Total time elapsed is 100 minutes (MP4)


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