Biology subjects

Lucks, J. B.

Publications and source records attributed to Lucks, J. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Design and characterization of peptide-responsive biosensors

Gram-positive bacteria use secreted peptides and peptide-responsive transcription factors for quorum sensing. Here, we demonstrate that intracellular expression of these peptide effectors with their cognate transcription factors leads to regulation of gene expression in both in vitro and in vivo systems. Because both effector and regulatory parts can be readily expressed, complex circuits, including auto-activation schemes, can be developed, with switch-like behaviors. Moreover, the peptide effectors can be fused to proteins, enabling a direct intracellular readout of translation and potentially allowing any protein to encode regulatory interactions in cells. Our results support a foundation for regulatory circuits that extend beyond canonical components such as LacI and TetR.

bioengineering↗

bioRxiv2026

Transcription-induced coacervation accelerates and sensitizes cell-free biosensing

Cell-free biosensors leverage in vitro gene expression reactions to detect chemicals. While inexpensive, modular, and distributable, these platforms are constrained by slow readouts at ambient temperatures, precluding practical field operation. In cells, phase separation accelerates biochemical reactions; however, recapitulating these gains in vitro has remained challenging for complex biochemistries. Here, we report the first self-assembling coacervate system that accelerates in vitro transcription. Prepared by simple mixing, coacervation with spermine and polyacrylic acid occurs dynamically in response to NTP consumption and co-localizes DNA templates and RNA polymerase to accelerate transcription, mimicking intracellular phenomena. We exploit this discovery to accelerate the cell-free biosensing of six ligands, demonstrating that coacervation can preserve platform modularity, improve sensitivity, retain lyophilization compatibility, function in field matrices, and reduce ambient-temperature time-to-signal by hours. This work contributes to a growing understanding of phase separation in biology and advances the use of membrane-less organization for real-world applications.

synthetic biology↗

bioRxiv2026

Design and discovery of "tug-of-war" riboswitches

Riboswitches are compact regulatory RNAs that modulate gene expression by switching between mutually exclusive structures in response to binding diverse signalling molecules. Riboswitches that regulate transcription have been shown to function through sequence overlap between their ligand binding aptamer domain and their regulatory expression platform, a constraint that limits their engineering for biotechnological applications. Here we describe a new, modular, transcriptional riboswitch architecture, termed "tug-of-war" (TOW), that removes this constraint, based on the discovery that ligand-stabilized RNA structures positioned immediately upstream of intrinsic terminators suppress termination. Using diverse aptamers, we create four distinct TOW riboswitches by pairing aptamer domains with terminators, and discover design principles for tuning their function. We demonstrate that upstream secondary and tertiary RNA structures generally inhibit intrinsic termination, consistent with a model of TOW antitermination dependant upon steric competition for occupancy of the RNA polymerase exit channel. Finally, we discover the TOW architecture to be present in nature through bioinformatic analysis and experimental validation, finding that [~]10% of natural ZTP riboswitches - nearly all in gram-positive bacteria - employ the TOW architecture. Together, these findings define a new mechanism of nascent RNA-mediated antitermination that may be useful for searching for riboswitches in other domains of life, and establish TOW riboswitches as a highly modular platform for engineering transcriptional RNA regulators.

biochemistry↗

bioRxiv2026

Efficient and scalable modelling of cotranscriptional RNA folding with deterministic and iterative RNA structure sampling

AO_SCPLOWBSTRACTC_SCPLOWRNA structure sampling is central to modelling RNA ensembles, yet stochastic sampling methods are non-exhaustive, scale poorly, and are biased towards low-free-energy structures, while current suboptimal folding approaches generate an unpredictable exponential number of structures. These limitations are particularly problematic for modelling cotranscriptional folding, where vectorial synthesis continuously reshapes the energy landscape during transcription, stabilising transient out-of-equilibrium structures. Here we introduce iterative sampling, a deterministic framework that enumerates unique RNA secondary structures in strict order of increasing free energy, enabling progressive and exhaustive exploration of the structure space up to an arbitrary stopping criterion. To implement this approach, we developed two scalable algorithms, iterative deepening and a persistent data structure approach, that incrementally traverse the expansion tree by evolving partial structures in place, avoiding redundant recomputation and fixed energy windows. Implemented in memerna, this approach achieves orders-of-magnitude speedups over existing tools (10x over ViennaRNA; 100x over RNAstructure). Integration within the sample-and-select framework (R2D2) improves structural diversity and identifies conformations with greater agreement with experimental data. Comprehensive sampling further enables direct comparison of equilibrium and cotranscriptionally restrained ensembles. Analysis of the resulting structural probability distributions uncovers kinetic traps and putative transcriptional pause sites, supporting an intuitive cotranscriptional folding mechanism in which local 3'-hairpin formation transiently stabilises upstream structure to delay large-scale rearrangement. Together, these results establish iterative sampling as a scalable and general framework for resolving out-of-equilibrium RNA cotranscriptional folding. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/720041v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@dee200org.highwire.dtl.DTLVardef@c7af12org.highwire.dtl.DTLVardef@1b6f54dorg.highwire.dtl.DTLVardef@a2c97a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

Ubiquitous low-energy RNA fluctuations and energetic coupling measured by chemical probing

RNA function is governed by RNA folding, but strategies for measuring RNA folding thermodynamics are limited, and fundamental questions such as the energy of base pair opening remain debated. Here we introduce Probing-Resolved Inference of Molecular Energetics (PRIME) to extract nucleotide-resolution RNA structural energetics from scalable chemical probing experiments. Applying PRIME to diverse RNAs, we find that RNA base pairs and tertiary interactions dynamically open with free energies of 0.5-3 kcal/mol, revealing that RNA nucleotides ubiquitously sample open conformations at biologically accessible energies. PRIME further resolves energetic coupling across RNAs, providing an energetic understanding of RNA structural dynamics and long-range coordination in RNA folding. PRIME represents a widely accessible strategy for interrogating RNA thermodynamics, enabling mechanistic understanding and engineering of RNA biology.

biophysics↗

bioRxiv2025

High-throughput functional profiling and evolutionary covariation analysis of entire riboswitch sequences

Riboswitches are useful models for revealing how some RNA molecules undergo dynamic rearrangements of their structures to perform cellular functions. A great deal is known about the structure of riboswitch ligand-binding aptamer domains through evolutionary sequence covariation analysis. However, covariation analysis has been more difficult to apply to riboswitch expression platforms given their large range in cellular functions, and their large sequence diversity. Here, we develop an approach to identify whole transcriptional riboswitch sequences starting from their conserved aptamer domains. We then generate covariation models for the entire riboswitch including the aptamer domain and the expression platform. The method consists of first bioinformatically extending identified aptamer domains to include downstream sequence that could contain an expression platform. Filtering is then performed using either a computational prediction algorithm to identify bacterial intrinsic terminator sequences in the expression platform, or a high-throughput functional assay that uses massive parallel oligo synthesis and next generation sequencing to characterize transcriptional termination of riboswitch candidates as a function of ligand. Filtered sequences are then used to develop full riboswitch sequence covariation models. We developed this approach in the context of the fluoride riboswitch, characterizing 1901 fluoride riboswitch sequences using our high-throughput assay. We find that the prediction filtering approach results in few false positives to identify novel, highly functional fluoride riboswitch variants. Finally, we employ the computational approach to develop covariation models of the ZTP, lysine, and TPP riboswitches and find covariation support for previously published rearrangement mechanisms. Overall, our method represents a new hybrid computational and high throughput experimental approach to characterize large numbers of riboswitch sequences and to generate new covariation models of complete riboswitch sequences, which should expand our understanding of riboswitch mechanism and the evolution of RNA structure dynamics.

biochemistry↗

bioRxiv2025

Active learning-guided optimization of cell-free biosensors for lead testing in drinking water

Point-of-use diagnostics based on allosteric transcription factors (aTFs) are promising tools for environmental monitoring and human health. However, biosensors relying on natural aTFs rarely exhibit the sensitivity and selectivity needed for real-world applications, and traditional directed evolution struggles to optimize multiple biosensor properties at once. To overcome these challenges, we develop a multi-objective, machine learning (ML)-guided cell-free gene expression workflow for engineering aTF-based biosensors. Our approach rapidly generates high-quality sequence-to-function data, which we transform into an augmented paired dataset to train an ML model using directional labels that capture how aTF mutations alter performance. We apply our workflow to engineer the aTF PbrR as a point-of-use diagnostic for lead contamination in water. We tune the sensitivity of PbrR to sense at the U.S. Environmental Protection Agency (EPA) action level for lead and modify the selectivity away from zinc, a common metal found in water supplies. Finally, we show that the engineered PbrR functions in freeze-dried cell-free reactions, enabling a diagnostic capable of detecting lead in drinking water down to [~]5.7 ppb. Our ML-driven, multi-objective framework--powered by directional tokens-- can generalize to other biosensors and proteins, accelerating the development of synthetic biology tools for biotechnology applications.

synthetic biology↗

bioRxiv2025

Design Principles for Polymerase Strand Recycling Circuits

Cell-free biosensing systems are being engineered as versatile and programmable diagnostic technologies. A core component of cell-free biosensors are programmable molecular circuits that improve biosensor speed, sensitivity and specificity by performing molecular computations such as logic evaluation and signal amplification. In previous work, we developed one such circuit system called Polymerase Strand Recycling (PSR) which amplifies cell-free molecular circuits by using T7 RNA polymerase off-target transcription to recycle nucleic acid inputs. We showed that PSR circuits can be configured to detect RNA target inputs as well as be interfaced with allosteric transcription factor-based biosensors to amplify signal and enhance sensitivity. Here we expand the development of PSR circuit design principles to generalize the platform for detecting a diverse set of model microRNA inputs. We show that PSR circuit function can be enhanced through engineering T7 RNAP, and present troubleshooting strategies to optimize PSR circuit performance.

synthetic biology↗

bioRxiv2024

Semi-automated Production of Cell-free Biosensors

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

synthetic biology↗

bioRxiv2024

Developing, characterizing and modelingCRISPR-based point-of-use pathogen diagnostics

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

synthetic biology↗

bioRxiv2024

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.

synthetic biology↗

bioRxiv2024

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.

biochemistry↗

bioRxiv2024

Ultra-sensitive water contaminant detection with transcription factor interfaced microcantilevers

Water contamination is a growing global concern, creating a need to develop technologies that can detect a range of target compounds at the required thresholds. Here, we address this need by merging biological allosteric transcription factors with DNA coated nanomechanical microcantilevers to detect chemicals in water with digital readout. After proof-of-concept demonstration and optimization to detect tetracycline with the TetR transcription factor, we use the CadC transcription factor to detect Pb2+ and Cd2+ in water at concentrations down to 2 ppb and 1 ppb, respectively, in less than fifteen minutes. A computational model suggests this improvement in sensitivity could be achieved by the DNA coated microcantilever surface changing transcription factor binding properties. Our findings demonstrate a promising new approach for water quality monitoring with fast, highly sensitive, digital readouts.

synthetic biology↗

bioRxiv2023

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 {degrees}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 {degrees}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.

molecular biology↗

bioRxiv2023

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.

synthetic biology↗

bioRxiv2022

Tuning Strand Displacement Kinetics Enables Programmable ZTP Riboswitch Dynamic Range in vivo

Recent 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 in vivo gene expression assays in E. coli, 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.

molecular biology↗

bioRxiv2022

Cotranscriptional RNA strand displacement 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 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 cotranscriptional strand displacement to mediate the switching mechanism. The coupling of transcription and translation in bacteria motivates the intriguing question as to whether translational riboswitches can utilize the same mechanistic features. Here we investigate this question by studying the Escherichia coli thiB thiamine 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, which based on sequence complementarity with the AD P1 stem suggested the possibility of an intermediate structure reminiscent of transcriptional riboswitches that exploit strand displacement. Point mutation analysis of this intermediate structure, followed by designed changes to P1, supported a strand displacement mechanism for E. coli thiB. This work provides an important new example of diverse riboswitch AD-EP combinations that exploit this switching mechanism.

molecular biology↗

bioRxiv2022

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 enteric bacteria 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 subunits. 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/493739v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@6a6281org.highwire.dtl.DTLVardef@6942f7org.highwire.dtl.DTLVardef@ab7ce8org.highwire.dtl.DTLVardef@957d55_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗