Biology subjects

Lucks, J. B.

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

36 records · Page 2Linked to original sources

bioRxiv2022

Bioengineering multifunctional extracellular vesicles for targeted delivery of biologics to T cells

Genetically modifying T cells can enable applications ranging from cancer immunotherapy to HIV treatment, yet delivery of T cell-targeted therapeutics remains challenging. Extracellular vesicles (EVs) are nanoscale particles secreted by all cells that naturally encapsulate and transfer proteins and nucleic acids, making them an attractive and clinically-relevant platform for engineering biocompatible delivery vehicles. We report a suite of technologies for genetically engineering cells to produce multifunctional EV vehicles--without employing chemical modifications that complicate biomanufacturing. We display high affinity targeting domains on the EV surface to achieve specific, efficient binding to T cells, identify a protein tag to confer active cargo loading into EVs, and display fusogenic glycoproteins to increase EV uptake and fusion with recipient cells. We demonstrate integration of these technologies by delivering Cas9-sgRNA complexes to edit primary human T cells. These approaches could enable targeting vesicles to a range of cells for the efficient delivery of cargo.

bioengineering↗

bioRxiv2022

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. By encapsulating these systems in synthetic membranes, we can reintroduce features of the cell membrane, including molecular containment and selective permeability, which together could significantly enhance sensing capabilities. Here, we demonstrate robust and tunable performance of an encapsulated fluoride riboswitch inside of lipid vesicles. The riboswitch remains active upon encapsulation in lipid vesicles and responds to fluoride added to the surrounding solution. We find the sensitivity of the encapsulated sensor can be tuned by varying membrane composition. We then show that encapsulation protects the sensor from degradation by the sample and use two types of genetically encoded outputs to detect fluoride in real-world samples. This work establishes the feasibility of vesicle-encapsulated cell-free systems to detect environmentally relevant small molecules.

synthetic biology↗

bioRxiv2021

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 aptamers P1 helix and the expression platforms 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.

biochemistry↗

bioRxiv2021

Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

Cell-free biosensors are emerging as powerful platforms for monitoring human and environmental health. Here, we expand the capabilities of biosensors by interfacing their outputs 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 biosensors with strand displacement circuits, show that these circuits allow fine-tuning of reaction kinetics and faster response times, and 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 target molecule being detected. We believe this work establishes a pathway to create "smart" diagnostics that use molecular computations to enhance the speed, robustness and utility of biosensors.

synthetic biology↗

bioRxiv2020

RNA sequence and structure determinants of Pol III transcriptional termination in human cells

The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that at the end of RNA transcripts, tracts comprised of multiple uracils are required for Pol III termination, whether upstream RNA secondary structure in the nascent transcript is necessary for robust transcriptional termination is still subject to debate. We sought to address this directly through the development of an in cellulo Pol III transcription termination assay using a synthetic biology approach. Specifically, we utilized the recently developed Tornado expression system and a stabilized Corn RNA aptamer to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. We found that in the absence of predicted secondary structure, only poly-U tracts longer than then the average length found in the human genome (4-5 nucleotides), efficiently terminate Pol III transcription. We found that shorter poly-U tracts could induce termination when placed in proximity to secondary structural elements, while secondary structure by itself was not sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.

molecular biology↗

bioRxiv2019

Chemical transcription roadblocking for nascent RNA display

Site-specific arrest of RNA polymerase is fundamental to several technologies that measure RNA structure and function. Current in vitro transcription roadblocking approaches inhibit transcription elongation using a protein blockade bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires the inclusion of a protein factor that is extrinsic to the minimal in vitro transcription reaction. In this work, we show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification efficiently and stably halts Escherichia coli RNA polymerase transcription. To facilitate diverse applications of chemical transcription roadblocking, we establish a simple and sequence-independent method for the preparation of internally modified double-stranded DNA templates by sequential PCR and translesion synthesis. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables nascent RNA molecules to be displayed from RNA polymerase in a minimal in vitro transcription reaction.

biochemistry↗

bioRxiv2019

Design and optimization of a cell-free atrazine biosensor

Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the component ratios of the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modularly designed, which should enable this work to serve as an effective proof-of-concept for rapid field-deployable detection of complex organic water contaminants.

synthetic biology↗

bioRxiv2019

Design of a transcriptional biosensor for the portable, on-demand detection of cyanuric acid

Rapid molecular biosensing is an emerging application area for synthetic biology. Here, we engineer a portable biosensor for cyanuric acid (CYA), an analyte of interest for human and environmental health, using a LysR-type transcription regulator (LTTR) from Pseudomonas within the context of Escherichia coli gene expression machinery. To overcome cross-host portability challenges of LTTRs, we rationally engineered hybrid Pseudomonas-E. coli promoters by integrating DNA elements required for transcriptional activity and ligand-dependent regulation from both hosts, which enabled E. coli to function as whole-cell biosensor for CYA. To alleviate challenges of whole-cell biosensing, we adapted these promoter designs to function within a freeze-dried E. coli cell-free system to sense CYA. This portable, on-demand system robustly detects CYA within an hour from laboratory and real-world samples and works with both fluorescent and colorimetric reporters. This work elucidates general principles to facilitate the engineering of a wider array of LTTR-based environmental sensors.

synthetic biology↗

bioRxiv2019

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

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 parts per million, the EPAs 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.

synthetic biology↗

bioRxiv2019

Dynamic control of pathway expression with riboregulated switchable feedback promoters

Dynamic pathway regulation has emerged as a promising strategy in metabolic engineering for improved system productivity and yield, and continues to grow in sophistication. Bacterial stress-response promoters allow dynamic gene regulation using the hosts natural transcriptional networks, but lack the flexibility to control the expression timing and overall magnitude of pathway genes. Here, we report a strategy that uses RNA transcriptional regulators to introduce another layer of control over the output of natural stress-response promoters. This new class of gene expression cassette, called a riboregulated switchable feedback promoter (rSFP), can be modularly activated using a variety of mechanisms, from manual induction to quorum sensing. We develop and apply rSFPs to regulate a toxic cytochrome P450 enzyme in the context of a Taxol precursor biosynthesis pathway and show this leads to 2.4x fold higher titers than from the best reported strain. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, protein and biologic production, and many other applications.

synthetic biology↗

bioRxiv2019

A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation

Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure and function by forming transient intermediate folds. This is especially true for transcriptional riboswitches in the 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 determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate a regulatory RNA strand displacement reaction and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for ZTP riboswitch antitermination and suggest general strategies by which nascent RNA molecules can navigate cotranscriptional folding pathways efficiently.

biochemistry↗

bioRxiv2018

De novo Design of Translational RNA Repressors

A central goal of synthetic biology is the development of methods for the predictable control of gene expression. RNA is an attractive substrate by which to achieve this goal because the relationship between its sequence, structure, and function is being uncovered with increasing depth. In addition, design approaches that use this relationship are becoming increasingly effective, as evidenced by significant progress in the de novo design of RNA-based gene regulatory mechanisms that activate transcription and translation in bacterial cells. However, the design of synthetic RNA mechanisms that are efficient and versatile repressors of gene expression has lagged, despite their importance for gene regulation and genetic circuit construction. We address this gap by developing two new classes of RNA regulators, toehold repressors and looped antisense oligonucleotides (LASOs), that repress translation of a downstream gene in response to an arbitrary input RNA sequence. Characterization studies show that these designed RNAs robustly repress translation, are highly orthogonal, and can be multiplexed with translational activators. We show that our LASO design can repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity and predictability. These results demonstrate significant yet easy-to-implement improvements in the design of synthetic RNA repressors for synthetic biology, and point more broadly to design principles for repressive RNA interactions relevant to modern drug design.

synthetic biology↗

bioRxiv2018

De-Novo-Designed Translational Repressors for Multi-Input Cellular Logic

Synthetic biology aims to apply engineering principles toward the development of novel biological systems for biotechnology and medicine. Despite efforts to expand the set of high-performing parts for genetic circuits, achieving more complex circuit functions has often been limited by the idiosyncratic nature and crosstalk of commonly utilized parts. Here, we present a molecular programming strategy that implements RNA-based repression of translation using de-novo-designed RNAs to realize high-performance orthogonal parts with mRNA detection and multi-input logic capabilities. These synthetic post-transcriptional regulators, termed toehold repressors and three-way junction (3WJ) repressors, efficiently suppress translation in response to cognate trigger RNAs with nearly arbitrary sequences using thermodynamically and kinetically favorable linear-linear RNA interactions. Automated in silico optimization of thermodynamic parameters yields improved toehold repressors with up to 300-fold repression, while in-cell SHAPE-Seq measurements of 3WJ repressors confirm their designed switching mechanism in living cells. Leveraging the absence of sequence constraints, we identify eight- and 15-component sets of toehold and 3WJ repressors, respectively, that provide high orthogonality. The modularity, wide dynamic range, and low crosstalk of the repressors enable their direct integration into ribocomputing devices that provide universal NAND and NOR logic capabilities and can perform multi-input RNA-based logic. We demonstrate these capabilities by implementing a four-input NAND gate and the expression NOT((A1 AND A2) OR (B1 AND B2)) in Escherichia coli. These features make toehold and 3WJ repressors important new classes of translational regulators for biotechnological applications.

synthetic biology↗

bioRxiv2018

PLANT-Dx: A Molecular Diagnostic for Point of Use Detection of Plant Pathogens

Synthetic biology based diagnostic technologies have improved upon gold standard diagnostic methodologies by decreasing cost, increasing accuracy, and enhancing portability. However there has been little effort in adapting these technologies towards applications related to point-of-use monitoring of plant and crop health. Here, we take a step towards this vision by developing an approach that couples isothermal amplification of specific plant pathogen genomic sequences with customizable synthetic RNA regulators that are designed to trigger the production of a colorimetric output in cell-free gene expression reactions. We demonstrate our system can sense viral derived sequences with high-sensitivity and specificity, and can be utilized to directly detect viruses from infected plant material. Furthermore, we demonstrate that the entire system can operate using only body heat and naked-eye visual analysis of outputs. We anticipate these strategies to be important components of user-friendly and deployable diagnostic systems that can be configured to detect a range of important plant pathogens.

synthetic biology↗

bioRxiv2018

DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data

MotivationRNA molecules can undergo complex structural dynamics, especially during transcription, which influence their biological functions. Recently developed high-throughput chemical probing experiments study RNA cotranscriptional folding to generate nucleotide-resolution reactivities for each length of a growing nascent RNA and reflect structural dynamics. However, the manual annotation and qualitative interpretation of reactivity across these large datasets can be nuanced, laborious, and difficult for new practitioners. We developed a quantitative and systematic approach to automatically detect RNA folding events from these datasets to reduce human bias/error, standardize event discovery, and generate hypotheses about RNA folding trajectories for further analysis and experimental validation.\n\nResultsDetection of Unknown Events with Tunable Thresholds (DUETT) identifies RNA structural transitions in cotranscriptional RNA chemical probing datasets. DUETT employs a feedback control-inspired method and a linear regression approach and relies on interpretable and independently tunable parameter thresholds to match qualitative user expectations with quantitatively identified folding events. We validate the approach by identifying known RNA structural transitions within the cotranscriptional folding pathways of the Escherichia coli signal recognition particle (SRP) RNA and the Bacillus cereus crcB fluoride riboswitch. We identify previously overlooked features of these datasets such as heightened reactivity patterns in the SRP RNA about 12 nucleotide lengths before base pair rearrangement. We then apply a sensitivity analysis to identify tradeoffs when choosing parameter thresholds. Finally, we show that DUETT is tunable across a wide range of contexts, enabling flexible application to study broad classes of RNA folding mechanisms.\n\nAvailabilityhttps://github.com/BagheriLab/DUETT\n\nContactjblucks@northwestern.edu, n-bagheri@northwestern.edu

bioinformatics↗

bioRxiv2018

Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry

Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial {sigma}70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

synthetic biology↗

bioRxiv2018

Computationally Reconstructing Cotranscriptional RNA Folding Pathways from Experimental Data Reveals Rearrangement of Non-Native Folding Intermediates

The series of RNA folding events that occur during transcription, or a cotranscriptional folding pathway, can critically influence the functional roles of RNA in the cell. Here we present a method, Reconstructing RNA Dynamics from Data (R2D2), to uncover details of cotranscriptional folding pathways by predicting RNA secondary and tertiary structures from cotranscriptional SHAPE-Seq data. We applied R2D2 to the folding of the Escherichia coli Signal Recognition Particle (SRP) RNA sequence and show that this sequence undergoes folding through non-native intermediate structures that require significant structural rearrangement before reaching the functional native structure. Secondary structure folding pathway predictions and all-atom molecular dynamics simulations of folding intermediates suggest that this rearrangement can proceed through a toehold mediated strand displacement mechanism, which can be disrupted and rescued with point mutations. These results demonstrate that even RNAs with simple functional folds can undergo complex folding processes during synthesis, and that small variations in their sequence can drastically affect their cotranscriptional folding pathways.\n\nHighlights- Computational methods predict RNA structures from cotranscriptional SHAPE-Seq data\n- The E. coli SRP RNA folds into non-native structural intermediates cotranscriptionally\n- These structures rearrange dynamically to form an extended functional fold\n- Point mutations can disrupt and rescue cotranscriptional RNA folding pathways

biochemistry↗

bioRxiv2018

Estimating RNA structure chemical probing reactivities from reverse transcriptase stops and mutations.

Chemical probing experiments interrogate RNA structures by creating covalent adducts on RNA molecules in structure-dependent patterns. Adduct positions are then detected through conversion of the modified RNAs into complementary DNA (cDNA) by reverse transcription (RT) as either stops (RT-stops) or mutations (RT-mutations). Statistical analysis of the frequencies of RT-stops and RT-mutations can then be used to estimate a measure of chemical probing reactivity at each nucleotide of an RNA, which reveals properties of the underlying RNA structure. Inspired by recent work that showed that different reverse transcriptase enzymes show distinct biases for detecting adducts as either RT-stops or RT-mutations, here we use a statistical modeling framework to derive an equation for chemical probing reactivity using experimental signatures from both RT-stops and RT-mutations within a single experiment. The resulting formula intuitively matches the expected result from considering reactivity to be defined as the fraction of adduct observed at each position in an RNA at the end of a chemical probing experiment. We discuss assumptions and implementation of the model, as well as ways in which the model may be experimentally validated.

biochemistry↗