This work presents a programmable molecular device that integrates Boolean logic computation with spatial confinement to overcome limitations in affinity, specificity, and off-target binding, and demonstrates a strategy for programming high-fidelity molecular interactions on interested cell surfaces.
Abstract
Achieving precise and robust cell-surface recognition in complex biological environments is challenging due to inherent trade-offs in affinity, specificity, and off-target binding. Herein, we present a programmable molecular device that integrates Boolean logic computation with spatial confinement to overcome these limitations. Our system employs valence-controllable, split DNAzyme modules assembled on a tetrahedral DNA nanostructure (TDN). The peroxidase-mimicking activity is stringently gated by a cell-surface AND logic, requiring the co-localization of two adjacent modules on target protein clusters for activation. This spatial constraint effectively eliminates stochastic or unintended signal leakage originating from solution-phase reactions or nontarget cells. Upon activation, the DNAzyme catalyzes the biotinylation of neighboring membrane protein clusters, generating stable multivalent adhesion sites. Quantitative dissociation kinetics reveal that the trivalent design of the DNAzyme modules promotes highly cooperative binding, resulting in uniform, long-lived complexes on target cells. We demonstrate that this approach enables specific recognition and highly efficient isolation of target cells from mixed cell populations and clinical samples, showcasing a strategy for programming high-fidelity molecular interactions on interested cell surfaces.
Cell membrane receptors are pivotal targets in precise therapeutics, yet their ubiquitous expression across tissues remains a fundamental barrier to achieving cell-specific intervention. To overcome the limitations of conventional monotargeting approaches, we developed a light-gated DNA nanoclaw machine (L-DNM) that integrates high-specificity recognition, spatiotemporally controlled therapy, and real-time monitoring of molecular activation within a unified nanoplatform. The L-DNM employed a multivalent targeting mechanism directed against a triple-marker signature, epithelial cell adhesion molecule (EpCAM), MUC1, and nucleolin (NCL), achieving exceptional targeting accuracy toward MCF-7 human breast cancer cells even in heterogeneous environments. Its novel photocleavable aptamer design ensures that the Met-inhibiting function remains biologically inert until activated by UV irradiation. This strategy enables precise spatiotemporal control over receptor tyrosine kinase (RTK) inhibition with minimal off-target effects. Furthermore, the system couples therapeutic activation with instantaneous electrochemiluminescence (ECL) reporting, transforming molecular recognition events into quantifiable signals with high signal-to-noise ratio in complex matrices. By unifying multiplexed targeting, light-gated activation, and self-reporting capability, the L-DNM platform represents a transformative shift from conventional therapeutics to adaptive, intelligent theranostic systems.
Cell-surface receptors integrate biochemical identity with mechanical information, yet methods for measuring receptor-specific force transmission across cell populations remain limited by the difficulty of coupling diverse recognition reagents to nucleic-acid tension probes. Here, we establish HUH endonuclease chemistry as a modular interface between antibodies and DNA- based mechanosensors. We develop two complementary strategies: genetically encoded HUH- antibody fusions that generate site-defined antibody-oligonucleotide conjugates in a single reaction, and a photocrosslinkable Protein G-HUH adaptor that enables covalent attachment of existing antibodies to DNA probes. Both approaches preserve antibody recognition while providing a programmable nucleic-acid handle for Rupture and Deliver Tension Gauge Tethers (RAD-TGTs). Using antibodies against beta1 integrin and HER2, we resolve receptor-specific mechanical phenotypes across cancer cell lines. Combining orthogonal features of beta1-integrin engagement and HER2 mechanical heterogeneity provides greater discrimination among cell types than either measurement alone, demonstrating that multidimensional mechanical phenotypes contain information not captured by individual force measurements. We further extend the platform to DNA:PNA tension probes to mitigate extracellular nuclease degradation and use antibody- functionalized RAD-TGTs to quantify force-dependent receptor engagement and pharmacological responses in immune cells. Together, these studies establish a modular antibody-to-nucleic-acid interface that expands DNA-based tension sensing beyond a restricted set of ligands and enables quantitative, multidimensional profiling of receptor-specific mechanical behavior across heterogeneous cell populations.
Matthew R. Pawlak, Lidia K. Limon, Andrew M. Baldys et al.· bioRxiv· 0 citations
A fluorescence chip based on a membrane-anchored, bipedal DNAzyme walker assembled on small extracellular vesicles (sEVs) surfaces via catalytic hairpin assembly (CHA) opens a new paradigm for designing efficient DNA nanomachines on membrane-enveloped targets.
Protein switches offer powerful strategies for detecting small molecules, yet current designs often suffer from limited dynamic range, lack of reversible control, and poor adaptability to nonfluorescent platforms. Here, we report a semisynthetic affinity-activated protein switch strategy that enables Signal-ON detection of small molecules across diverse environments, including live cells, Drosophila brain, and lateral flow assays. The switch consists of a self-labeling protein fused to a sensing protein and a synthetic probe containing a small-molecule ligand, a sterically shielded N′-3 urea nitrogen biotin derivative (N3B), and a tagging moiety. Ligand binding induces a conformational change that exposes the N3B for streptavidin-based signal output. This design achieves high dynamic range (up to 142-fold), low background, and nanomolar sensitivity for detecting sulfonamide, while demonstrating the transferability of the platform to trimethoprim detection. Reversible switching is enabled by the reduced N3B-streptavidin affinity, allowing signal reset via competitive displacement. In Drosophila brain, the protein switch enables spatially resolved ex vivo imaging of drug exposure. Furthermore, conjugation to gold nanoparticles allows robust Signal-ON colorimetric detection on lateral flow test strips. This work establishes a generalizable framework for building programmable protein switches with tunable output modes and potential diagnostic applicability.
First-generation LUNAR constructs can detect both oligonucleotides and plasmid double-stranded DNA with nanomolar sensitivity in mammalian cells and future work will focus on improving sensitivity, fold-change, and multiplexing capabilities for sequence-specific DNA detection.
Boao Xia, Nicholas A. Kalogriopoulos, Ruoxin Wen et al.· bioRxiv· 0 citations
