Single Cell Analysis of Proteoforms.

We introduce single cell Proteoform imaging Mass Spectrometry (scPiMS), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single cells dropcast onto glass slides. Sampling and detection of whole proteoforms by individual ion mass spectrometry enable a scalable approach to single cell proteomics. This new scPiMS platform addresses the throughput bottleneck in single cell proteomics and boosts the cell processing rate by several fold while accessing protein composition with higher coverage.

Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids.

The therapeutic potential of small interfering RNAs (siRNAs) is limited by their poor stability and low cellular uptake. When formulated as spherical nucleic acids (SNAs), siRNAs are resistant to nuclease degradation and enter cells without transfection agents with enhanced activity compared to their linear counterparts; however, the gene silencing activity of SNAs is limited by endosomal entrapment, a problem that impacts many siRNA-based nanoparticle constructs. To increase cytosolic delivery, SNAs are formulated using calcium chloride (CaCl2 ) instead of the conventionally used sodium chloride (NaCl). The divalent calcium (Ca2+ ) ions remain associated with the multivalent SNA and have a higher affinity for SNAs compared to their linear counterparts. Importantly, confocal microscopy studies show a 22% decrease in the accumulation of CaCl2 -salted SNAs within the late endosomes compared to NaCl-salted SNAs, indicating increased cytosolic delivery. Consistent with this finding, CaCl2 -salted SNAs comprised of siRNA and antisense DNA all exhibit enhanced gene silencing activity (up to 20-fold), compared to NaCl-salted SNAs regardless of sequence or cell line (U87-MG and SK-OV-3) studied. Moreover, CaCl2 -salted SNA-based forced intercalation probes show improved cytosolic mRNA detection.

Cellular Delivery of Large Functional Proteins and Protein-Nucleic Acid Constructs via Localized Electroporation.

Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (ß-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.

Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters.

We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ∼75-fold turn-on signal and limits of detection (LODs) as low as ∼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ∼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.

Implantable Fluorogenic DNA Biosensor for Stress Detection.

Implantable sensors that can monitor analytes related to cognitive and physiological status have gained significant focus in recent years. We have developed an implantable biosensor to detect dehydroepiandrosterone sulfate (DHEA-S), a biomarker related to stress. The biosensor strategy was based on the principle of forced intercalation (FIT) aptamers designed to detect subtle intramolecular changes during aptamer-target binding events. By incorporating a steroid-specific fluorogenic aptamer into a hydrogel, the sensitivity and biostability of the FIT biosensor fiber were improved, which were essential for designing implantable sensors to monitor biomarker levels in the living body. The polyethylenimine-based hydrogel chosen for this study produced an optically transparent cross-linked network with optimal microstructure, physicochemical, and mechanical properties, making it suitable for optical biosensors. The in vitro studies showed that the biosensor fiber was successfully activated in human serum and skin analogue, providing a linear response to physiological concentrations of the steroid. We believe that this type of implantable platform can be effective in monitoring more complex biomarkers associated with physiological or psychological health.

Spatially-Encoding Hydrogels With DNA to Control Cell Signaling.

Patterning biomolecules in synthetic hydrogels offers routes to visualize and learn how spatially-encoded cues modulate cell behavior (e.g., proliferation, differentiation, migration, and apoptosis). However, investigating the role of multiple, spatially defined biochemical cues within a single hydrogel matrix remains challenging because of the limited number of orthogonal bioconjugation reactions available for patterning. Herein, a method to pattern multiple oligonucleotide sequences in hydrogels using thiol-yne photochemistry is introduced. Rapid hydrogel photopatterning of hydrogels with micron resolution DNA features (≈1.5 µm) and control over DNA density are achieved over centimeter-scale areas using mask-free digital photolithography. Sequence-specific DNA interactions are then used to reversibly tether biomolecules to patterned regions, demonstrating chemical control over individual patterned domains. Last, localized cell signaling is shown using patterned protein-DNA conjugates to selectively activate cells on patterned areas. Overall, this work introduces a synthetic method to achieve multiplexed micron resolution patterns of biomolecules onto hydrogel scaffolds, providing a platform to study complex spatially-encoded cellular signaling environments.

Modular Nucleic Acid Scaffolds for Synthesizing Monodisperse and Sequence-Encoded Antibody Oligomers.

Synthesizing protein oligomers that contain exact numbers of multiple different proteins in defined architectures is challenging. DNA-DNA interactions can be used to program protein assembly into oligomers; however, existing methods require changes to DNA design to achieve different numbers and oligomeric sequences of proteins. Herein, we develop a modular DNA scaffold that uses only six synthetic oligonucleotides to organize proteins into defined oligomers. As a proof-of-concept, model proteins (antibodies) are oligomerized into dimers and trimers, where antibody function is retained. Illustrating the modularity of this technique, dimer and trimer building blocks are then assembled into pentamers containing three different antibodies in an exact stoichiometry and oligomeric sequence. In sum, this report describes a generalizable method for organizing proteins into monodisperse, sequence-encoded oligomers using DNA. This advance will enable studies into how oligomeric protein sequences affect material properties in areas spanning pharmaceutical development, cascade catalysis, synthetic photosynthesis, and membrane transport.