Engineering a cell-free biosensor signal amplification circuit with polymerase strand recycling.

Cell-free systems are powerful synthetic biology technologies because of their ability to recapitulate sensing and gene expression without the complications of living cells. Cell-free systems can perform even more advanced functions when genetic circuits are incorporated as information processing components. Here we expand cell-free biosensing by engineering a highly specific isothermal signal amplification circuit called polymerase strand recycling (PSR) that leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We develop design rules for PSR circuit components and use these rules to construct modular biosensors that can directly sense different RNA targets with limits of detection in the nM range and high specificity. We then use PSR for signal amplification within allosteric transcription factor-based biosensors for small molecule detection. We use a double equilibrium model of transcription factor:DNA and transcription factor:ligand binding interactions to predict biosensor sensitivity enhancement by PSR, and then demonstrate this approach experimentally by achieving 3.6-4.6-fold decreases in biosensor EC50 to sub micromolar ranges. We believe this work expands the current capabilities of cell-free circuits by incorporating PSR, which we anticipate will have a wide range of uses within biotechnology.

RNA folding kinetics control riboswitch sensitivity in vivo.

Riboswitches are ligand-responsive gene-regulatory RNA elements that perform key roles in maintaining cellular homeostasis. Understanding how riboswitch sensitivity is controlled is critical to understanding how highly conserved aptamer domains are deployed in a variety of contexts with different sensitivity demands. Here we uncover new roles by which RNA folding dynamics control riboswitch sensitivity in cells. By investigating the Clostridium beijerinckii pfl ZTP riboswitch, we identify multiple mechanistic routes of altering expression platform sequence and structure to slow RNA folding, all of which enhance riboswitch sensitivity. Applying these methods to riboswitches with diverse aptamer architectures that regulate transcription and translation with ON and OFF logic demonstrates the generality of our findings, indicating that any riboswitch that operates in a kinetic regime can be sensitized by slowing expression platform folding. Comparison of the most sensitized versions of these switches to equilibrium aptamer:ligand dissociation constants suggests a limit to the sensitivities achievable by kinetic RNA switches. Our results add to the growing suite of knowledge and approaches that can be used to rationally program cotranscriptional RNA folding for biotechnology applications, and suggest general RNA folding principles for understanding dynamic RNA systems in other areas of biology.

The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

Dynamic RNA synthetic biology: new principles, practices and potential.

An increased appreciation of the role of RNA dynamics in governing RNA function is ushering in a new wave of dynamic RNA synthetic biology. Here, we review recent advances in engineering dynamic RNA systems across the molecular, circuit and cellular scales for important societal-scale applications in environmental and human health, and bioproduction. For each scale, we introduce the core concepts of dynamic RNA folding and function at that scale, and then discuss technologies incorporating these concepts, covering new approaches to engineering riboswitches, ribozymes, RNA origami, RNA strand displacement circuits, biomaterials, biomolecular condensates, extracellular vesicles and synthetic cells. Considering the dynamic nature of RNA within the engineering design process promises to spark the next wave of innovation that will expand the scope and impact of RNA biotechnologies.

The Effect of Pseudoknot Base Pairing on Cotranscriptional Structural Switching of the Fluoride Riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of E. coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37 °C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65 °C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

Genetically encoding multiple functionalities into extracellular vesicles for the targeted delivery of biologics to T cells.

The genetic modification of T cells has advanced cellular immunotherapies, yet the delivery of biologics specifically to T cells remains challenging. Here we report a suite of methods for the genetic engineering of cells to produce extracellular vesicles (EVs)-which naturally encapsulate and transfer proteins and nucleic acids between cells-for the targeted delivery of biologics to T cells without the need for chemical modifications. Specifically, the engineered cells secreted EVs that actively loaded protein cargo via a protein tag and that displayed high-affinity T-cell-targeting domains and fusogenic glycoproteins. We validated the methods by engineering EVs that delivered Cas9-single-guide-RNA complexes to ablate the gene encoding the C-X-C chemokine co-receptor type 4 in primary human CD4+ T cells. The strategy is amenable to the targeted delivery of biologics to other cell types.

Teaching systematic, reproducible model development using synthetic biology.

We present an educational unit to teach computational modeling, a vital part of chemical engineering curricula, through the lens of synthetic biology. Lectures, code, and homework questions provide conceptual and practical introductions to each computational method involved in the model development process, along with perspectives on how methods can be iterated upon to arrive at a final model. Ultimately, this content can be applied broadly to address questions in synthetic biology and classical chemical engineering.

At-Home, Cell-Free Synthetic Biology Education Modules for Transcriptional Regulation and Environmental Water Quality Monitoring.

As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by simply adding water and DNA to freeze-dried crude extracts of non-pathogenic Escherichia coli. We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a "build-your-own" activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high-school students in their classrooms─and at home─without professional laboratory equipment. This work promises to catalyze access to interactive synthetic biology education opportunities.

A transient intermediate RNA structure underlies the regulatory function of the E. coli thiB TPP translational riboswitch.

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand binding through the coordinated action of a ligand-binding aptamer domain (AD) and a downstream expression platform (EP). Previous studies of transcriptional riboswitches have uncovered diverse examples that utilize structural intermediates that compete with the AD and EP folds to mediate the switching mechanism on the timescale of transcription. Here we investigate whether similar intermediates are important for riboswitches that control translation by studying the Escherichia coli thiB thiamin pyrophosphate (TPP) riboswitch. Using cellular gene expression assays, we first confirmed that the riboswitch acts at the level of translational regulation. Deletion mutagenesis showed the importance of the AD-EP linker sequence for riboswitch function. Sequence complementarity between the linker region and the AD P1 stem suggested the possibility of an intermediate nascent RNA structure called the antisequestering stem that could mediate the thiB switching mechanism. Experimentally informed secondary structure models of the thiB folding pathway generated from chemical probing of nascent thiB structures in stalled transcription elongation complexes confirmed the presence of the antisequestering stem, and showed it may form cotranscriptionally. Additional mutational analysis showed that mutations to the antisequestering stem break or bias thiB function according to whether the antisequestering stem or P1 is favored. This work provides an important example of intermediate structures that compete with AD and EP folds to implement riboswitch mechanisms.

Tuning strand displacement kinetics enables programmable ZTP riboswitch dynamic range in vivo.

A large body of work has shown that transcriptional riboswitches function through internal strand displacement mechanisms that guide the formation of alternative structures which drive regulatory outcomes. Here, we sought to investigate this phenomenon using the Clostridium beijerinckii pfl ZTP riboswitch as a model system. Using functional mutagenesis with Escherichia coli gene expression assays, we show that mutations designed to slow strand displacement of the expression platform enable precise tuning of riboswitch dynamic range (2.4-34-fold), depending on the type of kinetic barrier introduced, and the position of the barrier relative to the strand displacement nucleation site. We also show that expression platforms from a range of different Clostridium ZTP riboswitches contain sequences that impose these barriers to affect dynamic range in these different contexts. Finally, we use sequence design to flip the regulatory logic of the riboswitch to create a transcriptional OFF-switch, and show that the same barriers to strand displacement tune dynamic range in this synthetic context. Together, our findings further elucidate how strand displacement can be manipulated to alter the riboswitch decision landscape, suggesting that this could be a mechanism by which evolution tunes riboswitch sequence, and providing an approach to optimize synthetic riboswitches for biotechnology applications.

The accuracy and usability of point-of-use fluoride biosensors in rural Kenya.

Geogenic fluoride contaminates the water of tens of millions of people. However, many are unaware of the fluoride content due in part to shortcomings of detection methods. Biosensor tests are a relatively new approach to water quality testing that address many of these shortcomings but have never been tested by non-experts in a "real-world" setting. We therefore sought to assess the accuracy and usability of a point-of-use fluoride biosensor using surveys and field tests in Nakuru County, Kenya. Biosensor tests accurately classified elevated fluoride (≥1.5 ppm) in 89.5% of the 57 samples tested. Usability was also high; all participants were able to use the test and correctly interpreted all but one sample. These data suggest that biosensor tests can provide accurate, meaningful water quality data that help non-experts make decisions about the water they consume. Further scaling of these technologies could provide new approaches to track global progress towards Sustainable Development Goal 6.

Robust and tunable performance of a cell-free biosensor encapsulated in lipid vesicles.

Cell-free systems have enabled the development of genetically encoded biosensors to detect a range of environmental and biological targets. Encapsulation of these systems in synthetic membranes to form artificial cells can reintroduce features of the cellular membrane, including molecular containment and selective permeability, to modulate cell-free sensing capabilities. Here, we demonstrate robust and tunable performance of a transcriptionally regulated, cell-free riboswitch encapsulated in lipid membranes, allowing the detection of fluoride, an environmentally important molecule. Sensor response can be tuned by varying membrane composition, and encapsulation protects from sensor degradation, facilitating detection in real-world samples. These sensors can detect fluoride using two types of genetically encoded outputs, enabling detection of fluoride at the Environmental Protection Agency maximum contaminant level of 0.2 millimolars. This work demonstrates the capacity of bilayer membranes to confer tunable permeability to encapsulated, genetically encoded sensors and establishes the feasibility of artificial cell platforms to detect environmentally relevant small molecules.

At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring.

As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by just-adding-water and DNA to freeze-dried crude extracts of Escherichia coli . We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a 'build-your-own' activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high school students in their classrooms - and at home - without professional laboratory equipment or researcher oversight. This work promises to catalyze access to interactive synthetic biology education opportunities.

Zur and zinc increase expression of E. coli ribosomal protein L31 through RNA-mediated repression of the repressor L31p.

Bacteria can adapt in response to numerous stress conditions. One such stress condition is zinc depletion. The zinc-sensing transcription factor Zur regulates the way numerous bacterial species respond to severe changes in zinc availability. Under zinc sufficient conditions, Zn-loaded Zur (Zn2-Zur) is well-known to repress transcription of genes encoding zinc uptake transporters and paralogues of a few ribosomal proteins. Here, we report the discovery and mechanistic basis for the ability of Zur to up-regulate expression of the ribosomal protein L31 in response to zinc in E. coli. Through genetic mutations and reporter gene assays, we find that Zur achieves the up-regulation of L31 through a double repression cascade by which Zur first represses the transcription of L31p, a zinc-lacking paralogue of L31, which in turn represses the translation of L31. Mutational analyses show that translational repression by L31p requires an RNA hairpin structure within the l31 mRNA and involves the N-terminus of the L31p protein. This work uncovers a new genetic network that allows bacteria to respond to host-induced nutrient limiting conditions through a sophisticated ribosomal protein switching mechanism.

Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing.

The detection of chemicals using natural allosteric transcription factors is a powerful strategy for point-of-use molecular sensing, particularly using fieldable cell-free gene expression (CFE) systems. However, the reliance of detection schemes on characterized protein-based sensors limits the number of measurable analytes. One alternative solution to this issue is to develop new sensors by generating RNA aptamers against the target analyte and then incorporating them directly into a riboswitch scaffold for ligand-inducible genetic control of a reporter protein. However, this strategy has not generated more than a handful of successful portable cell-free molecular sensors. To address this gap, here we convert dopamine-binding aptamers into functional dopamine-sensing riboswitches that regulate gene expression in a freeze-dried CFE reaction. We then develop an assay for direct detection and semi-quantification of dopamine in human urine. We anticipate that this work will be broadly applicable for converting many in vitro-generated RNA aptamers into fieldable molecular diagnostics.

How does RNA fold dynamically?

Recent advances in interrogating RNA folding dynamics have shown the classical model of RNA folding to be incomplete. Here, we pose three prominent questions for the field that are at the forefront of our understanding of the importance of RNA folding dynamics for RNA function. The first centers on the most appropriate biophysical framework to describe changes to the RNA folding energy landscape that a growing RNA chain encounters during transcriptional elongation. The second focuses on the potential ubiquity of strand displacement - a process by which RNA can rapidly change conformations - and how this process may be generally present in broad classes of seemingly different RNAs. The third raises questions about the potential importance and roles of cellular protein factors in RNA conformational switching. Answers to these questions will greatly improve our fundamental knowledge of RNA folding and function, drive biotechnological advances that utilize engineered RNAs, and potentially point to new areas of biology yet to be discovered.

Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch.

RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamer's P1 helix and the expression platform's transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.

Riboswitches have challenged our understanding of biological regulation for almost two decades. The ability of small molecules to bind to RNA and control gene expression offers another layer of regulation and the potential for direct action by compounds in the environment. While some riboswitches have been well studied, we lack a general understanding of how changes in RNA structure switch genetic expression from "On" to "Off". In this study, the authors propose an elegant "strand displacement" model to explain how the RNA structure shifts between "On" and "Off" states as the concentration of small molecule ligand changes. These observations help us to understand how riboswitches enable genetic decision-making. The data provide a possible general mechanism for understanding how the competition between different strand displacement outcomes can influence RNA folding. Understanding RNA folding pathways could advance the successful design of drugs that target RNA.

Programming cell-free biosensors with DNA strand displacement circuits.

Cell-free biosensors are powerful platforms for monitoring human and environmental health. Here, we expand their capabilities by interfacing them with toehold-mediated strand displacement circuits, a dynamic DNA nanotechnology that enables molecular computation through programmable interactions between nucleic acid strands. We develop design rules for interfacing a small molecule sensing platform called ROSALIND with toehold-mediated strand displacement to construct hybrid RNA-DNA circuits that allow fine-tuning of reaction kinetics. We use these design rules to build 12 different circuits that implement a range of logic functions (NOT, OR, AND, IMPLY, NOR, NIMPLY, NAND). Finally, we demonstrate a circuit that acts like an analog-to-digital converter to create a series of binary outputs that encode the concentration range of the molecule being detected. We believe this work establishes a pathway to create 'smart' diagnostics that use molecular computations to enhance the speed and utility of biosensors.

ROSALIND: Rapid Detection of Chemical Contaminants with In Vitro Transcription Factor-Based Biosensors.

ROSALIND (RNA Output Sensors Activated by Ligand Induction) is an in vitro biosensing system that detects small molecules using regulated transcription reactions. It consists of three key components: (1) RNA polymerases, (2) allosteric protein transcription factors, and (3) synthetic DNA transcription templates that together regulate the synthesis of a fluorescence-activating RNA aptamer. The system can detect a wide range of chemicals including antibiotics, small molecules, and metal ions. We have demonstrated that ROSALIND can be lyophilized and transported at ambient conditions for water testing on-site. Here, we describe how to set up a ROSALIND reaction for detecting various chemical contaminants in water using a model transcription factor as well as how to build a new ROSALIND sensor.