Alexander A. Green

Lipid membrane‐bound synthetic cells provide programmable, cell‐like compartments for on‐demand biomolecule production. However, most cell‐free gene expression systems in synthetic cells are regulated by user‐imposed cues rather than signals associated with environmental or physiological states. Here, we present an acidity‐transducing synthetic cell that converts external pH changes into nucleic acid information to drive in situ protein synthesis. The system integrates gramicidin A proton channels for pH sensing, triplex‐forming single‐stranded DNA (ssDNA) that releases a trigger ssDNA upon acidification, and a toehold switch RNA that activates translation in response to the released trigger ssDNA. This work further reveals that tuning the annealing length between the pH‐responsive and trigger ssDNAs controls the trigger‐release pH, which is critical for enabling acid‐triggered protein synthesis. The synthetic cells retain their pH‐responsive activity when embedded in alginate hydrogels, creating acidity‐responsive materials that synthesize proteins in situ rather than release preloaded cargo. Cell‐penetrating peptide tagging further enables selective protein release and target‐specific binding. This work establishes a molecular transduction strategy for programming synthetic cell materials to sense environmentally relevant acidity and generates functional biomolecules on demand.

Rapid, portable detection of multiple nucleic acid targets is essential for infectious disease surveillance and precision oncology. CRISPR-based diagnostics have set a high bar for sensitivity and single-base specificity, yet their reliance on collateral nuclease activity complicates multiplexing, integration with amplification, and point-of-care deployment. Here, we present TIMBER (templated incision mediated by endonucleases of repair), a non-CRISPR platform that achieves comparable performance without nonspecific nuclease activity. TIMBER uses a repair endonuclease to cleave probes containing abasic sites only when they are hybridized to a matched target. The system exhibits strong specificity, enabling single-nucleotide polymorphism discrimination. TIMBER provides an analytical limit of detection of 12 pM without preamplification or one copy per μL (1.6 aM) with preamplification through RT-PCR or RT-LAMP, with results observable by visual fluorescence or on lateral flow. We deploy TIMBER to detect SARS-CoV-2 in clinical saliva samples and further demonstrate multiplex detection of four targets, enabling identification of EGFR mutations in lung cancer samples. This approach offers a flexible, rapid, and low-instrument-ready solution for diverse nucleic acid diagnostics, from viral detection to cancer genotyping.

Conventional methods for monitoring toxic heavy metals typically require sophisticated laboratory instrumentation and leave a critical gap for rapid, on-site detection. Herein we present Transcription-factor-Occluded Nick Extension or TONE, a rapid, isothermal biosensing platform for heavy metal detection that exploits allosteric transcription factor (aTF) regulation to gate DNA strand-displacement amplification. In this approach, operator sequences modified with deoxyinosine (dI) substitutions are employed. When bound by an aTF, the dI sites are shielded from cleavage by endonuclease V. Upon exposure to target heavy metals, the aTF dissociates, permitting the enzyme to nick the DNA and trigger a strand-displacement reaction. The amplified DNA is then detected via an instrument-free lateral flow assay, delivering a visual readout within 25 min at room temperature. We demonstrate the utility of TONE using the TetR aTF and further adapt the assay for copper and lead detection through the transcription factors CsoR and CadC, respectively, reliably detecting concentrations as low as 40 and 80 nM, respectively. TONE offers a sensitive, instrument-free assay that operates at room temperature, with the potential for field deployment, for use in environmental monitoring and other applications requiring rapid heavy metal analysis.

Cell-free synthetic biology offers a rapid prototyping environment for molecular diagnostics, yet the utility of these systems is often limited by the slow kinetics and restricted multiplexing capabilities of traditional protein reporters. Here, we report the BINOCULAR (BIspecific Novel Orthogonal Coiled-coil-Using LAteral flow Reporter) system based on supramolecular coiled-coil peptide conjugates that dramatically accelerates signal generation and enables robust multiplexing in cell-free transcription-translation reactions. Using BINOCULARs as toehold switch outputs, we achieve visible readout times of less than 5 min in lateral flow assays, achieving a ≥12-fold increase in speed compared to standard cell-free reporters like β-galactosidase or fluorescent proteins. To demonstrate the utility of this platform, we integrated the bispecific reporters with loop-mediated isothermal amplification and lateral flow assays for the detection of mycoplasma, a common adventitious agent in biomanufacturing. The integrated system demonstrates exceptional sensitivity, achieving a detection limit of 50 genomic copies/mL for mycoplasma and a mammalian internal control. Cell-free assay results were available within 5 min following a 20 min amplification step, and we successfully achieved the simultaneous detection of three distinct targets in a single reaction. Crucially, the platform maintains reliable performance in complex matrices and after lyophilization, making it more accessible for point-of-use applications. Our findings suggest that the modular BINOCULAR architecture can overcome current bottlenecks in cell-free sensing systems, providing a versatile and scalable framework for rapid, point-of-use screening across clinical and industrial applications.

CRISPR–Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans-cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans-cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features and building on emerging protospacer adjacent motif (PAM)-independent Cas12a diagnostics that use engineered DNA activators and split-guide architectures, we introduce RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans-cleavage activity matching conventional Cas12a. We then apply RAPID to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-independent RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR for samples with Ct ≤ 33.

CRISPR-Cas12a nucleases are classically activated through CRISPR RNA (crRNA)-guided and PAM-dependent target recognition, which together establish a canonical heteroduplex associated with nuclease activation. Here we identify a crRNA– and PAM-independent activation pathway for Cas12a that reveals previously unrecognized conformational plasticity within its nucleic acid–recognition interface. We show that short RNAs can directly occupy the canonical crRNA–binding channel and trigger a catalytically competent trans cleavage state in the absence of PAM recognition or canonical R-loop formation. Biochemical assays indicate that short RNAs bind the crRNA–binding channel and are competitively displaced by cognate crRNA, consistent with binding at a conserved nucleic acid–binding interface. Cryo-electron microscopy (cryo-EM) further reveals that Cas12a maintains its global catalytic architecture while exhibiting loss of canonical PAM-dependent stabilization and increased flexibility of the RuvC lid, alongside accommodation of a noncanonical RNA–DNA hybrid with inverted polarity relative to the crRNA–target duplex. This crRNA-independent activation pathway enables programmable and amplification-free detection of DNA and RNA targets independent of canonical guide-mediated recognition. Together, these findings define an alternative activation geometry for Cas12a and expand models of Class 2 CRISPR-Cas effector activation beyond crRNA– and PAM-directed recognition.

RNA-based sensors offer powerful and programmable control of gene expression, yet our understanding of the structural principles that govern their potential design space remains incomplete. Here, we challenged a community of designers to generate novel riboregulators capable of activating translation in response to specific RNA targets. Participants submitted diverse sequence architectures, which were synthesized and evaluated in a cell-free transcription-translation system. Across 100 designs, community-generated riboregulators displayed wide variability in activation dynamics, fold change, and structural features, outperforming some canonical toehold-switch designs and achieving up to 80-fold activation. Structural ensemble analyses identified accessibility patterns near the ribosome binding site that distinguish high- from low-performing regulators, highlighting the central role of RBS sequestration and release in modulating expression. Together, we demonstrate community-driven design can expand the accessible structural space of riboregulators and uncover mechanistic features governing translational activation. Our findings establish quantitative links between RNA folding energetics and gene expression output, providing design principles for next-generation programmable RNA sensors.

Rapid, portable detection of multiple nucleic acid targets is essential for infectious disease surveillance and precision oncology. CRISPR-based diagnostics have set a high bar for sensitivity and single-base specificity, yet their reliance on collateral nuclease activity complicates multiplexing, integration with amplification, and point-of-care deployment. Here we present TIMBER (Templated Incision Mediated By Endonucleases of Repair), a non-CRISPR, isothermal platform that achieves comparable performance without nonspecific nuclease activity. TIMBER uses a repair endonuclease to cleave probes containing abasic sites only when they are hybridized to a matched target. The system exhibits strong specificity, enabling single-nucleotide polymorphism discrimination. TIMBER provides an analytical limit of detection 12 pM without preamplification or 1 copy per µL (1.6 aM) with preamplification through RT-PCR or RT-LAMP, with results observable by visual fluorescence or on lateral flow. We deploy TIMBER to detect SARS-CoV-2 in clinical saliva samples and further demonstrate multiplex detection of four targets enabling identification of EGFR mutations in lung cancer samples. This approach offers a flexible, rapid, and low-instrument ready solution for diverse nucleic acid diagnostics, from viral detection to cancer genotyping. Graphical

Conventional methods for monitoring toxic heavy metals typically require sophisticated laboratory instrumentation and leave a critical gap for rapid, on-site detection. Herein we present Transcription-factor-Occluded Nick Extension or TONE, a rapid, isothermal biosensing platform for heavy metal detection that exploits allosteric transcription factor (aTF) regulation to gate DNA strand-displacement amplification. In this approach, operator sequences modified with deoxyinosine (dI) substitutions are employed. When bound by an aTF, the dI sites are shielded from cleavage by Endonuclease V. Upon exposure to target heavy metals, the aTF dissociates, permitting the enzyme to nick the DNA and trigger a strand-displacement reaction. The amplified DNA is then detected via an instrument-free lateral flow assay, delivering a visual readout within 25 minutes at room temperature. We demonstrate the utility of TONE using the TetR aTF and further adapt the assay for copper and lead detection through the transcription factors CsoR and CadC, respectively, achieving detection limits as low as 40 nM and 80 nM, respectively. TONE offers a sensitive, low-cost, and field-deployable solution for environmental monitoring and other applications requiring rapid heavy metal analysis.