Kevin Yehl

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.

Nanoscale optomechanical actuators for controlling mechanotransduction in living cells.

To control receptor tension optically at the cell surface, we developed an approach involving optomechanical actuator nanoparticles that are controlled with near-infrared light. Illumination leads to particle collapse, delivering piconewton forces to specific cell surface receptors with high spatial and temporal resolution. We demonstrate optomechanical actuation by controlling integrin-based focal adhesion formation, cell protrusion and migration, and T cell receptor activation.

High-speed DNA-based rolling motors powered by RNase H

DNA-based machines that walk by converting chemical energy into controlled motion could be of use in applications such as next generation sensors, drug delivery platforms, and biological computing. Despite their exquisite programmability, DNA-based walkers are, however, challenging to work with due to their low fidelity and slow rates (~1 nm/min). Here, we report DNA-based machines that roll rather than walk, and consequently have a maximum speed and processivity that is three-orders of magnitude greater than conventional DNA motors. The motors are made from DNA-coated spherical particles that hybridise to a surface modified with complementary RNA; motion is achieved through the addition of RNase H, which selectively hydrolyses hybridised RNA. Spherical motors move in a self-avoiding manner, whereas anisotropic particles, such as dimerised particles or rod-shaped particles travel linearly without a track or external force. Finally, we demonstrate detection of single nucleotide polymorphism by measuring particle displacement using a smartphone camera.

Quantum dots encapsulated within phospholipid membranes: phase-dependent structure, photostability, and site-selective functionalization.

Lipid vesicle encapsulation is an efficient approach to transfer quantum dots (QDs) into aqueous solutions, which is important for renewable energy applications and biological imaging. However, little is known about the molecular organization at the interface between a QD and lipid membrane. To address this issue, we investigated the properties of 3.0 nm CdSe QDs encapsulated within phospholipid membranes displaying a range of phase transition temperatures (Tm). Theoretical and experimental results indicate that the QD locally alters membrane structure, and in turn, the physical state (phase) of the membrane controls the optical and chemical properties of the QDs. Using photoluminescence, ICP-MS, optical microscopy, and ligand exchange studies, we found that the Tm of the membrane controls optical and chemical properties of lipid vesicle-embedded QDs. Importantly, QDs encapsulated within gel-phase membranes were ultrastable, providing the most photostable non-core/shell QDs in aqueous solution reported to date. Atomistic molecular dynamics simulations support these observations and indicate that membranes are locally disordered displaying greater disordered organization near the particle-solution interface. Using this asymmetry in membrane organization near the particle, we identify a new approach for site-selective modification of QDs by specifically functionalizing the QD surface facing the outer lipid leaflet to generate gold nanoparticle-QD assemblies programmed by Watson-Crick base-pairing.

Knockdown of TNF-α by DNAzyme gold nanoparticles as an anti-inflammatory therapy for myocardial infarction.

In this study, we used deoxyribozyme (DNAzyme) functionalized gold nanoparticles (AuNPs) to catalytically silence tumor necrosis factor-α (TNF-α) in vivo as a potential therapeutic for myocardial infarction (MI). Using primary macrophages as a model, we demonstrated 50% knockdown of TNF-α, which was not attainable using Lipofectamine-based approaches. Local injection of DNAzyme conjugated to gold particles (AuNPs) in the rat myocardium yielded TNF-α knockdown efficiencies of 50%, which resulted in significant anti-inflammatory effects and improvement in acute cardiac function following MI. Our results represent the first example showing the use of DNAzyme AuNP conjugates in vivo for viable delivery and gene regulation. This is significant as TNF-α is a multibillion dollar drug target implicated in many inflammatory-mediated disorders, thus underscoring the potential impact of DNAzyme-conjugated AuNPs.

Mechanically Induced Catalytic Amplification Reaction for Readout of Receptor-Mediated Cellular Forces

Mechanics play a fundamental role in cell biology, but detecting piconewton (pN) forces is challenging because of a lack of accessible and high throughput assays. A mechanically induced catalytic amplification reaction (MCR) for readout of receptor-mediated forces in cells is described. Mechanically labile DNA duplexes presenting ligands are surface immobilized such that specific receptor forces denature the duplex and thus expose a blocked primer. Amplification of primers is achieved using an isothermal polymerization reaction and quantified by fluorescence readout. As a proof of concept, the assay was used to test the activity of a mechanomodulatory drug and integrin adhesion receptor antibodies. To the best of our knowledge, this is the first example of a catalytic reaction triggered in response to molecular piconewton forces. The MCR may transform the field of mechanobiology by providing a new facile tool to detect receptor specific mechanics with the convenience of the polymerase chain reaction (PCR).

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.