Thermal-plex: fluidic-free, rapid sequential multiplexed imaging with DNA-encoded thermal channels.

Multiplexed fluorescence imaging is typically limited to three- to five-plex on standard setups. Sequential imaging methods based on iterative labeling and imaging enable practical higher multiplexing, but generally require a complex fluidic setup with several rounds of slow buffer exchange (tens of minutes to an hour for each exchange step). We report the thermal-plex method, which removes complex and slow buffer exchange steps and provides fluidic-free, rapid sequential imaging. Thermal-plex uses simple DNA probes that are engineered to fluoresce sequentially when, and only when, activated with transient exposure to heating spikes at designated temperatures (thermal channels). Channel switching is fast (<30 s) and is achieved with a commercially available and affordable on-scope heating device. We demonstrate 15-plex RNA imaging (five thermal × three fluorescence channels) in fixed cells and retina tissues in less than 4 min, without using buffer exchange or fluidics. Thermal-plex introduces a new labeling method for efficient sequential multiplexed imaging.

Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.

DNA probes that store mechanical information reveal transient piconewton forces applied by T cells.

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

Massively Parallelized Molecular Force Manipulation with On-Demand Thermal and Optical Control.

In single-molecule force spectroscopy (SMFS), a tethered molecule is stretched using a specialized instrument to study how macromolecules extend under force. One problem in SMFS is the serial and slow nature of the measurements, performed one molecule at a time. To address this long-standing challenge, we report on the origami polymer force clamp (OPFC) which enables parallelized manipulation of the mechanical forces experienced by molecules without the need for dedicated SMFS instruments or surface tethering. The OPFC positions target molecules between a rigid nanoscale DNA origami beam and a responsive polymer particle that shrinks on demand. As a proof-of-concept, we record the steady state and time-resolved mechanical unfolding dynamics of DNA hairpins using the fluorescence signal from ensembles of molecules and confirm our conclusion using modeling.

Engineering DNA-Functionalized Nanostructures to Bind Nucleic Acid Targets Heteromultivalently with Enhanced Avidity.

Improving the affinity of nucleic acids to their complements is an important goal for many fields spanning from genomics to antisense therapy and diagnostics. One potential approach to achieving this goal is to use multivalent binding, which often boosts the affinity between ligands and receptors, as exemplified by virus-cell binding and antibody-antigen interactions. Herein, we investigate the binding of heteromultivalent DNA-nanoparticle conjugates, where multiple unique oligonucleotides displayed on a nanoparticle form a multivalent complex with a long DNA target containing the complementary sequences. By developing a strategy to spatially pattern oligonucleotides on a nanoparticle, we demonstrate that the molecular organization of heteromultivalent nanostructures is critical for effective binding; patterned particles have a ∼23 order-of-magnitude improvement in affinity compared to chemically identical particles patterned incorrectly. We envision that nanostructures presenting spatially patterned heteromultivalent DNA will offer important biomedical applications given the utility of DNA-functionalized nanostructures in diagnostics and therapeutics.

Tunable DNA Origami Motors Translocate Ballistically Over µm Distances at nm/s Speeds.

Inspired by biological motor proteins, that efficiently convert chemical fuel to unidirectional motion, there has been considerable interest in developing synthetic analogues. Among the synthetic motors created thus far, DNA motors that undertake discrete steps on RNA tracks have shown the greatest promise. Nonetheless, DNA nanomotors lack intrinsic directionality, are low speed and take a limited number of steps prior to stalling or dissociation. Herein, we report the first example of a highly tunable DNA origami motor that moves linearly over micron distances at an average speed of 40 nm/min. Importantly, nanomotors move unidirectionally without intervention through an external force field or a patterned track. Because DNA origami enables precise testing of nanoscale structure-function relationships, we were able to experimentally study the role of motor shape, chassis flexibility, leg distribution, and total number of legs in tuning performance. An anisotropic rigid chassis coupled with a high density of legs maximizes nanomotor speed and endurance.

Seeded Heteroepitaxial Growth of Crystallizable Collagen Triple Helices: Engineering Multifunctional Two-Dimensional Core-Shell Nanostructures.

Engineering free-standing 2D nanomaterials with compositional, spatial, and functional control across size regimes from the nano- to mesoscale represents a significant challenge. Herein, we demonstrate a straightforward strategy for the thermodynamically controlled fabrication of multicomponent sectored nanosheets in which each sector can be chemically and spatially addressed independently and orthogonally. Collagen triple helices, comprising collagen-mimetic peptides (CMPs), are employed as molecularly programmable crystallizable units. Modulating their thermodynamic stability affords the controlled synthesis of 2D core-shell nanostructures via thermally driven heteroepitaxial growth. Structural information, gathered from SAXS and cryo-TEM, reveals that the distinct peptide domains maintain their intrinsic lattice structure and illuminates various mechanisms employed by CMP triple helices to alleviate the elastic strain associated with the interfacial lattice mismatch. Finally, we demonstrate that different sectors of the sheet surface can be selectively functionalized using bioorthogonal conjugation chemistry. Altogether, we establish a robust platform for constructing multifunctional 2D nanoarchitectures in which one can systematically program their compositional, spatial, and functional properties, which is a significant step toward their deployment into functional nanoscale devices.

Light-Responsive Polymer Particles as Force Clamps for the Mechanical Unfolding of Target Molecules.

Single-molecule force spectroscopy techniques are powerful tools for investigating the mechanical unfolding of biomolecules. However, they are limited in throughput and require dedicated instrumentation. Here, we report a force-generating particle that can unfold target molecules on-demand. The particle consists of a plasmonic nanorod core encapsulated with a thermoresponsive polymer shell. Optical heating of the nanorod leads to rapid collapse of the polymer, thus transducing light into mechanical work to unfold target molecules. The illumination tunes the duration and degree of particle collapse, thus controlling the lifetime and magnitude of applied forces. Single-molecule fluorescence imaging showed reproducible mechanical unfolding of DNA hairpins. We also demonstrate the triggering of 50 different particles in <1 min, exceeding the speed of conventional atomic force microscopy. The polymer force clamp represents a facile and bottom-up approach to force manipulation.

Ratiometric Tension Probes for Mapping Receptor Forces and Clustering at Intermembrane Junctions.

Short-range communication between cells is required for the survival of multicellular organisms. One mechanism of chemical signaling between adjacent cells employs surface displayed ligands and receptors that only bind when two cells make physical contact. Ligand-receptor complexes that form at the cell-cell junction and physically bridge two cells likely experience mechanical forces. A fundamental challenge in this area pertains to mapping the mechanical forces experienced by ligand-receptor complexes within such a fluid intermembrane junction. Herein, we describe the development of ratiometric tension probes for direct imaging of receptor tension, clustering, and lateral transport within a model cell-cell junction. These probes employ two fluorescent reporters that quantify both the ligand density and the ligand tension and thus generate a tension signal independent of clustering. As a proof-of-concept, we applied the ratiometric tension probes to map the forces experienced by the T-cell receptor (TCR) during activation and showed the first direct evidence that the TCR-ligand complex experiences sustained pN forces within a fluid membrane junction. We envision that the ratiometric tension probes will be broadly useful for investigating mechanotransduction in juxtacrine signaling pathways.

Revealing nanostructures in brain tissue via protein decrowding by iterative expansion microscopy.

Many crowded biomolecular structures in cells and tissues are inaccessible to labelling antibodies. To understand how proteins within these structures are arranged with nanoscale precision therefore requires that these structures be decrowded before labelling. Here we show that an iterative variant of expansion microscopy (the permeation of cells and tissues by a swellable hydrogel followed by isotropic hydrogel expansion, to allow for enhanced imaging resolution with ordinary microscopes) enables the imaging of nanostructures in expanded yet otherwise intact tissues at a resolution of about 20 nm. The method, which we named 'expansion revealing' and validated with DNA-probe-based super-resolution microscopy, involves gel-anchoring reagents and the embedding, expansion and re-embedding of the sample in homogeneous swellable hydrogels. Expansion revealing enabled us to use confocal microscopy to image the alignment of pre-synaptic calcium channels with post-synaptic scaffolding proteins in intact brain circuits, and to uncover periodic amyloid nanoclusters containing ion-channel proteins in brain tissue from a mouse model of Alzheimer's disease. Expansion revealing will enable the further discovery of previously unseen nanostructures within cells and tissues.

Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope.

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.

DNA Gold Nanoparticle Motors Demonstrate Processive Motion with Bursts of Speed Up to 50 nm Per Second.

Synthetic motors that consume chemical energy to produce mechanical work offer potential applications in many fields that span from computing to drug delivery and diagnostics. Among the various synthetic motors studied thus far, DNA-based machines offer the greatest programmability and have shown the ability to translocate micrometer-distances in an autonomous manner. DNA motors move by employing a burnt-bridge Brownian ratchet mechanism, where the DNA "legs" hybridize and then destroy complementary nucleic acids immobilized on a surface. We have previously shown that highly multivalent DNA motors that roll offer improved performance compared to bipedal walkers. Here, we use DNA-gold nanoparticle conjugates to investigate and enhance DNA nanomotor performance. Specifically, we tune structural parameters such as DNA leg density, leg span, and nanoparticle anisotropy as well as buffer conditions to enhance motor performance. Both modeling and experiments demonstrate that increasing DNA leg density boosts the speed and processivity of motors, whereas DNA leg span increases processivity and directionality. By taking advantage of label-free imaging of nanomotors, we also uncover Lévy-type motion where motors exhibit bursts of translocation that are punctuated with transient stalling. Dimerized particles also demonstrate more ballistic trajectories confirming a rolling mechanism. Our work shows the fundamental properties that control DNA motor performance and demonstrates optimized motors that can travel multiple micrometers within minutes with speeds of up to 50 nm/s. The performance of these nanoscale motors approaches that of motor proteins that travel at speeds of 100-1000 nm/s, and hence this work can be important in developing protocellular systems as well next generation sensors and diagnostics.

Mechanical Stimulation of Adhesion Receptors Using Light-Responsive Nanoparticle Actuators Enhances Myogenesis.

The application of cyclic strain is known to enhance myoblast differentiation and muscle growth in vitro and in vivo. However, current techniques apply strain to full tissues or cell monolayers, making it difficult to evaluate whether mechanical stimulation at the subcellular or single-cell scales would drive myoblast differentiation. Here, we report the use of optomechanical actuator (OMA) particles, comprised of a ∼0.6 µm responsive hydrogel coating a gold nanorod (100 × 20 nm) core, to mechanically stimulate the integrin receptors in myoblasts. When illuminated with near-infrared (NIR) light, OMA nanoparticles rapidly collapse, exerting mechanical forces to cell receptors bound to immobilized particles. Using a pulsed illumination pattern, we applied cyclic integrin forces to C2C12 myoblasts cultured on a monolayer of OMA particles and then measured the cellular response. We found that 20 min of OMA actuation resulted in cellular elongation in the direction of the stimulus and enhancement of nuclear YAP1 accumulation, an effector of ERK phosphorylation. Cellular response was dependent on direct conjugation of RGD peptides to the OMA particles. Repeated OMA mechanical stimulation for 5 days led to enhanced myogenesis as quantified using cell alignment, fusion, and sarcomeric myosin expression in myotubes. OMA-mediated myogenesis was sensitive to the geometry of stimulation but not to MEK1/2 inhibition. Finally, we found that OMA stimulation in regions proximal to the nucleus resulted in localization of the transcription activator YAP-1 to the nucleus, further suggesting the role of YAP1 in mechanotransduction in C2C12 cells. These findings demonstrate OMAs as a novel tool for studying the role of spatially localized forces in influencing myogenesis.

Localized Nanoscale Heating Leads to Ultrafast Hydrogel Volume-Phase Transition.

The rate of the volume-phase transition for stimuli-responsive hydrogel particles ranging in size from millimeters to nanometers is limited by the rate of water transport, which is proportional to the surface area of the particle. Here, we hypothesized that the rate of volume-phase transition could be accelerated if the stimulus is geometrically controlled from the inside out, thus facilitating outward water ejection. To test this concept, we applied transient absorption spectroscopy, laser temperature-jump spectroscopy, and finite-element analysis modeling to characterize the dynamics of the volume-phase transition of hydrogel particles with a gold nanorod core. Our results demonstrate that the nanoscale heating of the hydrogel particle core led to an ultrafast, 60 ns particle collapse, which is 2-3 orders of magnitude faster than the response generated from conventional heating. This is the fastest recorded response time of a hydrogel material, thus opening potential applications for such stimuli-responsive materials.

Molecular Tension Probes to Investigate the Mechanopharmacology of Single Cells: A Step toward Personalized Mechanomedicine.

Given that dysregulation of mechanics contributes to diseases ranging from cancer metastasis to lung disease, it is important to develop methods for screening the efficacy of drugs that target cellular forces. Here, nanoparticle-based tension sensors are used to quantify the mechanical response of individual cells upon drug treatment. As a proof-of-concept, the activity of bronchodilators is tested on human airway smooth muscle cells derived from seven donors, four of which are asthmatic. It is revealed that airway smooth muscle cells isolated from asthmatic donors exhibit greater traction forces compared to the control donors. Additionally, the mechanical signal is abolished using myosin inhibitors or further enhanced in the presence of inflammatory inducers, such as nicotine. Using the signal generated by the probes, single-cell dose-response measurements are performed to determine the "mechano" effective concentration (mechano-EC50 ) of albuterol, a bronchodilator, which reduces integrin forces by 50%. Mechano-EC50 values for each donor present discrete readings that are differentially enhanced as a function of nicotine treatment. Importantly, donor mechano-EC50 values varied by orders of magnitude, suggesting significant variability in their sensitivity to nicotine and albuterol treatment. To the best of the authors' knowledge, this is the first study harnessing a piconewton tension sensor platform for mechanopharmacology.