Bidirectional linkage of DNA barcodes for the multiplexed mapping of higher-order protein interactions in cells.

Capturing the full complexity of the diverse hierarchical interactions in the protein interactome is challenging. Here we report a DNA-barcoding method for the multiplexed mapping of pairwise and higher-order protein interactions and their dynamics within cells. The method leverages antibodies conjugated with barcoded DNA strands that can bidirectionally hybridize and covalently link to linearize closely spaced interactions within individual 3D protein complexes, encoding and decoding the protein constituents and the interactions among them. By mapping protein interactions in cancer cells and normal cells, we found that tumour cells exhibit a larger diversity and abundance of protein complexes with higher-order interactions. In biopsies of human breast-cancer tissue, the method accurately identified the cancer subtype and revealed that higher-order protein interactions are associated with cancer aggressiveness.

Accessible detection of SARS-CoV-2 through molecular nanostructures and automated microfluidics.

Accurate and accessible nucleic acid diagnostics is critical to reducing the spread of COVID-19 and resuming socioeconomic activities. Here, we present an integrated platform for the direct detection of SARS-CoV-2 RNA targets near patients. Termed electrochemical system integrating reconfigurable enzyme-DNA nanostructures (eSIREN), the technology leverages responsive molecular nanostructures and automated microfluidics to seamlessly transduce target-induced molecular activation into an enhanced electrochemical signal. Through responsive enzyme-DNA nanostructures, the technology establishes a molecular circuitry that directly recognizes specific RNA targets and catalytically enhances signaling; only upon target hybridization, the molecular nanostructures activate to liberate strong enzymatic activity and initiate cascading reactions. Through automated microfluidics, the system coordinates and interfaces the molecular circuitry with embedded electronics; its pressure actuation and liquid-guiding structures improve not only analytical performance but also automated implementation. The developed platform establishes a detection limit of 7 copies of RNA target per µl, operates against the complex biological background of native patient samples, and is completed in <20 min at room temperature. When clinically evaluated, the technology demonstrates accurate detection in extracted RNA samples and direct swab lysates to diagnose COVID-19 patients.

Collaborative Equilibrium Coupling of Catalytic DNA Nanostructures Enables Programmable Detection of SARS-CoV-2.

Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID-19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS-CoV-2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme-DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi-enzyme catalysis; through system-wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single-copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS-CoV-2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID-19 patients.

Extracellular vesicle drug occupancy enables real-time monitoring of targeted cancer therapy.

Current technologies to measure drug-target interactions require complex processing and invasive tissue biopsies, limiting their clinical utility for cancer treatment monitoring. Here we develop an analytical platform that leverages circulating extracellular vesicles (EVs) for activity-based assessment of tumour-specific drug-target interactions in patient blood samples. The technology, termed extracellular vesicle monitoring of small-molecule chemical occupancy and protein expression (ExoSCOPE), utilizes bio-orthogonal probe amplification and spatial patterning of molecular reactions within matched plasmonic nanoring resonators to achieve in situ analysis of EV drug dynamics. It measures changes in drug occupancy and protein composition in molecular subpopulations of EVs. When used to monitor various targeted therapies, the ExoSCOPE revealed EV signatures that closely reflected cellular treatment efficacy. We further applied the technology for clinical cancer diagnostics and treatment monitoring. Using a small volume of blood, the ExoSCOPE accurately classified disease status and rapidly distinguished between targeted treatment outcomes, within 24 h after treatment initiation.

Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2.

Despite the importance of nucleic acid testing in managing the COVID-19 pandemic, current detection approaches remain limited due to their high complexity and extensive processing. Here, we describe a molecular nanotechnology that enables direct and sensitive detection of viral RNA targets in native clinical samples. The technology, termed catalytic amplification by transition-state molecular switch (CATCH), leverages DNA-enzyme hybrid complexes to form a molecular switch. By ratiometric tuning of its constituents, the multicomponent molecular switch is prepared in a hyperresponsive state-the transition state-that can be readily activated upon the binding of sparse RNA targets to turn on substantial enzymatic activity. CATCH thus achieves superior performance (~8 RNA copies/µl), direct fluorescence detection that bypasses all steps of PCR (<1 hour at room temperature), and versatile implementation (high-throughput 96-well format and portable microfluidic assay). When applied for clinical COVID-19 diagnostics, CATCH demonstrated direct and accurate detection in minimally processed patient swab samples.

Exosome-templated nanoplasmonics for multiparametric molecular profiling.

Exosomes are nanoscale vesicles distinguished by characteristic biophysical and biomolecular features; current analytical approaches, however, remain univariate. Here, we develop a dedicated platform for multiparametric exosome analysis-through simultaneous biophysical and biomolecular evaluation of the same vesicles-directly in clinical biofluids. Termed templated plasmonics for exosomes, the technology leverages in situ growth of gold nanoshells on vesicles to achieve multiselectivity. For biophysical selectivity, the nanoshell formation is templated by and tuned to distinguish exosome dimensions. For biomolecular selectivity, the nanoshell plasmonics locally quenches fluorescent probes only if they are target-bound on the same vesicle. The technology thus achieves multiplexed analysis of diverse exosomal biomarkers (e.g., proteins and microRNAs) but remains unresponsive to nonvesicle biomarkers. When implemented on a microfluidic, smartphone-based sensor, the platform is rapid, sensitive, and wash-free. It not only distinguished biomarker organizational states in native clinical samples but also showed that the exosomal subpopulation could more accurately differentiate patient prognosis.

Barcoded DNA nanostructures for the multiplexed profiling of subcellular protein distribution.

Massively parallel DNA sequencing is established, yet high-throughput protein profiling remains challenging. Here, we report a barcoding approach that leverages the combinatorial sequence content and the configurational programmability of DNA nanostructures for high-throughput multiplexed profiling of the subcellular expression and distribution of proteins in whole cells. The barcodes are formed by in situ hybridization of tetrahedral DNA nanostructures and short DNA sequences conjugated with protein-targeting antibodies, and by nanostructure-assisted ligation (either enzymatic or chemical) of the nanostructures and exogenous DNA sequences bound to nanoparticles of different sizes (which cause these localization sequences to differentially distribute across subcellular compartments). Compared with linear DNA barcoding, the nanostructured barcodes enhance the signal by more than 100-fold. By implementing the barcoding approach on a microfluidic device for the analysis of rare patient samples, we show that molecular subtypes of breast cancer can be accurately classified and that subcellular spatial markers of disease aggressiveness can be identified.

Subtyping of circulating exosome-bound amyloid ß reflects brain plaque deposition.

Despite intense interests in developing blood measurements of Alzheimer's disease (AD), the progress has been confounded by limited sensitivity and poor correlation to brain pathology. Here, we present a dedicated analytical platform for measuring different populations of circulating amyloid ß (Aß) proteins - exosome-bound vs. unbound - directly from blood. The technology, termed amplified plasmonic exosome (APEX), leverages in situ enzymatic conversion of localized optical deposits and double-layered plasmonic nanostructures to enable sensitive, multiplexed population analysis. It demonstrates superior sensitivity (~200 exosomes), and enables diverse target co-localization in exosomes. Employing the platform, we find that prefibrillar Aß aggregates preferentially bind with exosomes. We thus define a population of Aß as exosome-bound (Aß42+ CD63+) and measure its abundance directly from AD and control blood samples. As compared to the unbound or total circulating Aß, the exosome-bound Aß measurement could better reflect PET imaging of brain amyloid plaques and differentiate various clinical groups.

Microhexagon gradient array directs spatial diversification of spinal motor neurons.

Motor neuron diversification and regionalization are important hallmarks of spinal cord development and rely on fine spatiotemporal release of molecular cues. Here, we present a dedicated platform to engineer complex molecular profiles for directed neuronal differentiation. Methods: The technology, termed microhexagon interlace for generation of versatile and fine gradients (microHIVE), leverages on an interlocking honeycomb lattice of microstructures to dynamically pattern molecular profiles at a high spatial resolution. By packing the microhexagons as a divergent, mirrored array, the platform not only enables maximal mixing efficiency but also maintains a small device footprint. Results: Employing the microHIVE platform, we developed optimized profiles of growth factors to induce rostral-caudal patterning of spinal motor neurons, and directed stem cell differentiation in situ into a spatial continuum of different motor neuron subtypes. Conclusions: The differentiated cells showed progressive RNA and protein signatures, consistent with that of representative brachial, thoracic and lumbar regions of the human spinal cord. The microHIVE platform can thus be utilized to develop advanced biomimetic systems for the study of diseases in vitro.

Visual and modular detection of pathogen nucleic acids with enzyme-DNA molecular complexes.

Rapid, visual detection of pathogen nucleic acids has broad applications in infection management. Here we present a modular detection platform, termed enzyme-assisted nanocomplexes for visual identification of nucleic acids (enVision). The system consists of an integrated circuit of enzyme-DNA nanostructures, which function as independent recognition and signaling elements, for direct and versatile detection of pathogen nucleic acids from infected cells. The built-in enzymatic cascades produce a rapid color readout for the naked eye; the assay is thus fast (<2 h), sensitive (<10 amol), and readily quantified with smartphones. When implemented on a configurable microfluidic platform, the technology demonstrates superior programmability to perform versatile computations, for detecting diverse pathogen targets and their virus-host genome integration loci. We further design the enVision platform for molecular-typing of infections in patient endocervical samples. The technology not only improves the clinical inter-subtype differentiation, but also expands the intra-subtype coverage to identify previously undetectable infections.

Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis.

Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6-9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA) constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi) against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that can act as potent and highly specific male contraceptives.