Tension Sensing Nanoparticles for Mechano-Imaging at the Living/Nonliving Interface

Studying chemomechanical coupling at interfaces is important for fields ranging from lubrication and tribology to microfluidics and cell biology. Several polymeric macro- and microscopic systems and cantilevers have been developed to image forces at interfaces, but few materials are amenable for molecular tension sensing. To address this issue, we have developed a gold nanoparticle sensor for molecular tension-based fluorescence micro­scopy. As a proof of concept, we imaged the tension exerted by integrin receptors at the interface between living cells and a substrate with high spatial (<1 μm) resolution, at 100 ms acquisition times and with molecular specificity. We report integrin tension values ranging from 1 to 15 pN and a mean of ∼1 pN within focal adhesions. Through the use of a conventional fluorescence microscope, this method demonstrates a force sensitivity that is 3 orders of magnitude greater than is achievable by traction force microscopy or polydimethylsiloxane micropost arrays, which are the standard in cellular biomechanics

Tension Sensing Nanoparticles for Mechano-Imaging at the Living/Nonliving Interface

Studying chemomechanical coupling at interfaces is important for fields ranging from lubrication and tribology to microfluidics and cell biology. Several polymeric macro- and microscopic systems and cantilevers have been developed to image forces at interfaces, but few materials are amenable for molecular tension sensing. To address this issue, we have developed a gold nanoparticle sensor for molecular tension-based fluorescence micro­scopy. As a proof of concept, we imaged the tension exerted by integrin receptors at the interface between living cells and a substrate with high spatial (<1 μm) resolution, at 100 ms acquisition times and with molecular specificity. We report integrin tension values ranging from 1 to 15 pN and a mean of ∼1 pN within focal adhesions. Through the use of a conventional fluorescence microscope, this method demonstrates a force sensitivity that is 3 orders of magnitude greater than is achievable by traction force microscopy or polydimethylsiloxane micropost arrays, which are the standard in cellular biomechanics

Adhesive Dynamics Simulations of Highly Polyvalent DNA Motors

Molecular motors, such as myosin and kinesin, perform diverse tasks ranging from vesical transport to bulk muscle contraction. Synthetic molecular motors may eventually be harnessed to perform similar tasks in versatile synthetic systems. The most promising type of synthetic molecular motor, the DNA walker, can undergo processive motion but generally exhibits low speeds and virtually no capacity for force generation. However, we recently showed that highly polyvalent DNA motors (HPDMs) can rival biological motors by translocating at micrometer per minute speeds and generating 100+ pN of force. Accordingly, DNA nanotechnology-based designs may hold promise for the creation of synthetic, force-generating nanomotors. However, the dependencies of HPDM speed and force on tunable design parameters are poorly understood and difficult to characterize experimentally. To overcome this challenge, we present RoloSim, an adhesive dynamics software package for fine-grained simulations of HPDM translocation. RoloSim uses biophysical models for DNA duplex formation and dissociation kinetics to explicitly model tens of thousands of molecular scale interactions. These molecular interactions are then used to calculate the nano- and microscale motions of the motor. We use RoloSim to uncover how motor force and speed scale with several tunable motor properties such as motor size and DNA duplex length. Our results support our previously defined hypothesis that force scales linearly with polyvalency. We also demonstrate that HPDMs can be steered with external force, and we provide design parameters for novel HPDM-based molecular sensor and nanomachine designs

Titin-Based Nanoparticle Tension Sensors Map High-Magnitude Integrin Forces within Focal Adhesions

Mechanical forces transmitted through integrin transmembrane receptors play important roles in a variety of cellular processes ranging from cell development to tumorigenesis. Despite the importance of mechanics in integrin function, the magnitude of integrin forces within adhesions remains unclear. Literature suggests a range from 1 to 50 pN, but the upper limit of integrin forces remains unknown. Herein we challenge integrins with the most mechanically stable molecular tension probe, which is comprised of the immunoglobulin 27th (I27) domain of cardiac titin flanked with a fluorophore and gold nanoparticle. Cell experiments show that integrin forces unfold the I27 domain, suggesting that integrin forces exceed ∼30–40 pN. The addition of a disulfide bridge within I27 “clamps” the probe and resists mechanical unfolding. Importantly, incubation with a reducing agent initiates SH exchange, thus unclamping I27 at a rate that is dependent on the applied force. By recording the rate of S–S reduction in clamped I27, we infer that integrins apply 110 ± 9 pN within focal adhesions of rat embryonic fibroblasts. The rates of S–S exchange are heterogeneous and integrin subtype-dependent. Nanoparticle titin tension sensors along with kinetic analysis of unfolding demonstrate that a subset of integrins apply tension many fold greater than previously reported

An Endosomal Escape Trojan Horse Platform to Improve Cytosolic Delivery of Nucleic Acids

Endocytosis is a major bottleneck toward cytosolic delivery of nucleic acids, as the vast majority of nucleic acid drugs remain trapped within endosomes. Current trends to overcome endosomal entrapment and subsequent degradation provide varied success; however, active delivery agents such as cell-penetrating peptides have emerged as a prominent strategy to improve cytosolic delivery. Yet, these membrane-active agents have poor selectivity for endosomal membranes, leading to toxicity. A hallmark of endosomes is their acidic environment, which aids in degradation of foreign materials. Here, we develop a pH-triggered spherical nucleic acid that provides smart antisense oligonucleotide (ASO) release upon endosomal acidification and selective membrane disruption, termed DNA EndosomaL Escape Vehicle Response (DELVR). We anchor i-Motif DNA to a nanoparticle (AuNP), where the complement strand contains both an ASO sequence and a functionalized endosomal escape peptide (EEP). By orienting the EEP toward the AuNP core, the EEP is inactive until it is released through acidification-induced i-Motif folding. In this study, we characterize a small library of i-Motif duplexes to develop a structure-switching nucleic acid sequence triggered by endosomal acidification. We evaluate antisense efficacy using HIF1a, a hypoxic indicator upregulated in many cancers, and demonstrate dose-dependent activity through RT-qPCR. We show that DELVR significantly improves ASO efficacy in vitro. Finally, we use fluorescence lifetime imaging and activity measurement to show that DELVR benefits synergistically from nuclease- and pH-driven release strategies with increased ASO endosomal escape efficiency. Overall, this study develops a modular platform that improves the cytosolic delivery of nucleic acid therapeutics and offers key insights for overcoming intracellular barriers

A General Approach for Generating Fluorescent Probes to Visualize Piconewton Forces at the Cell Surface

Mechanical forces between cells and their extracellular matrix (ECM) are mediated by dozens of different receptors. These biophysical interactions play fundamental roles in processes ranging from cellular development to tumor progression. However, mapping the spatial and temporal dynamics of tension among various receptor–ligand pairs remains a significant challenge. To address this issue, we have developed a synthetic strategy to generate modular tension probes combining the native chemical ligation (NCL) reaction with solid phase peptide synthesis (SPPS). In principle, this approach accommodates virtually any peptide or expressed protein amenable to NCL. We generated a small library of tension probes displaying different ligands, flexible linkers, and fluorescent reporters, enabling the mapping of integrin and cadherin tension, and demonstrating the first example of long-term (∼3 days) molecular tension imaging. This approach provides a toolset to better understand mechanotransduction events fundamental to cell biology

A General Approach for Generating Fluorescent Probes to Visualize Piconewton Forces at the Cell Surface

Mechanical forces between cells and their extracellular matrix (ECM) are mediated by dozens of different receptors. These biophysical interactions play fundamental roles in processes ranging from cellular development to tumor progression. However, mapping the spatial and temporal dynamics of tension among various receptor–ligand pairs remains a significant challenge. To address this issue, we have developed a synthetic strategy to generate modular tension probes combining the native chemical ligation (NCL) reaction with solid phase peptide synthesis (SPPS). In principle, this approach accommodates virtually any peptide or expressed protein amenable to NCL. We generated a small library of tension probes displaying different ligands, flexible linkers, and fluorescent reporters, enabling the mapping of integrin and cadherin tension, and demonstrating the first example of long-term (∼3 days) molecular tension imaging. This approach provides a toolset to better understand mechanotransduction events fundamental to cell biology

Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem–loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells

Catalytic Deoxyribozyme-Modified Nanoparticles for RNAi-Independent Gene Regulation

DNAzymes are catalytic oligonucleotides with important applications in gene regulation, DNA computing, responsive soft materials, and ultrasensitive metal-ion sensing. The most significant challenge for using DNAzymes <i>in vivo</i> pertains to nontoxic delivery and maintaining function inside cells. We synthesized multivalent deoxyribozyme “10-23” gold nanoparticle (DzNP) conjugates, varying DNA density, linker length, enzyme orientation, and linker composition in order to study the role of the steric environment and gold surface chemistry on catalysis. DNAzyme catalytic efficiency was modulated by steric packing and proximity of the active loop to the gold surface. Importantly, the 10-23 DNAzyme was asymmetrically sensitive to the gold surface and when anchored through the 5′ terminus was inhibited 32-fold. This property was used to generate DNAzymes whose catalytic activity is triggered by thiol displacement reactions or by photoexcitation at λ = 532 nm. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease degradation, readily enter mammalian cells, and catalytically down-regulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for <i>in vivo</i> work

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