Resonator nanophotonic standing-wave array trap for single-molecule manipulation and measurement

Nanophotonic tweezers represent emerging platforms with significant potential for parallel manipulation and measurements of single biological molecules on-chip. However, trapping force generation represents a substantial obstacle for their broader utility. Here, we present a resonator nanophotonic standing-wave array trap (resonator-nSWAT) that demonstrates significant force enhancement. This platform integrates a critically-coupled resonator design to the nSWAT and incorporates a novel trap reset scheme. The nSWAT can now perform standard single-molecule experiments, including stretching DNA molecules to measure their force-extension relations, unzipping DNA molecules, and disrupting and mapping protein-DNA interactions. These experiments have realized trapping forces on the order of 20 pN while demonstrating base-pair resolution with measurements performed on multiple molecules in parallel. Thus, the resonator-nSWAT platform now meets the benchmarks of a table-top precision optical trapping instrument in terms of force generation and resolution. This represents the first demonstration of a nanophotonic platform for such single-molecule experiments.

Highly sensitive force measurements in an optically generated, harmonic hydrodynamic trap

The use of optical tweezers to measure forces acting upon microscopic particles has revolutionised fields from material science to cell biology. However, despite optical control capabilities, this technology is highly constrained by the material properties of the probe, and its use may be limited due to concerns about the effect on biological processes. Here we present a novel, optically controlled trapping method based on light-induced hydrodynamic flows. Specifically, we leverage optical control capabilities to convert a translationally invariant topological defect of a flow field into an attractor for colloids in an effectively one-dimensional harmonic, yet freely rotatable system. Circumventing the need to stabilise particle dynamics along an unstable axis, this novel trap closely resembles the isotropic dynamics of optical tweezers. Using magnetic beads, we explicitly show the existence of a linear force-extension relationship that can be used to detect femtoNewton-range forces with sensitivity close to the thermal limit. Our force measurements remove the need for laser-particle contact, while also lifting material constraints, which renders them a particularly interesting tool for the life sciences and engineering.

Chromatin fiber breaks into clutches under tension and crowding

The arrangement of nucleosomes inside chromatin is of extensive interest. While in vitro experiments have revealed the formation of 30 nm fibers, most in vivo studies have failed to confirm their presence in cell nuclei. To reconcile the diverging experimental findings, we characterized chromatin organization using a near atomistic model. The computed force-extension curve matches well with measurements from single-molecule experiments. Notably, we found that a dodeca-nucleosome in the two-helix zigzag conformation breaks into structures with nucleosome clutches and a mix of trimers and tetramers under tension. Such unfolded configurations can also be stabilized through trans interactions with other chromatin chains. Our study supports a hypothesis that disordered, in vivo chromatin configurations arise as folding intermediates from regular fibril structures. We further revealed that chromatin segments with fibril or clutch structures engaged in distinct binding modes and discussed the implications of these inter-chain interactions for a potential sol-gel phase transition.

Force Dependence of Proteins’ Transition State Position and the Bell–Evans Model

Single-molecule force spectroscopy has opened a new field of research in molecular biophysics and biochemistry. Pulling experiments on individual proteins permit us to monitor conformational transitions with high temporal resolution and measure their free energy landscape. The force–extension curves of single proteins often present large hysteresis, with unfolding forces that are higher than refolding ones. Therefore, the high energy of the transition state (TS) in these molecules precludes kinetic rates measurements in equilibrium hopping experiments. In irreversible pulling experiments, force-dependent kinetic rates measurements show a systematic discrepancy between the sum of the folding and unfolding TS distances derived by the kinetic Bell–Evans model and the full molecular extension predicted by elastic models. Here, we show that this discrepancy originates from the force-induced movement of TS. Specifically, we investigate the highly kinetically stable protein barnase, using pulling experiments and the Bell–Evans model to characterize the position of its kinetic barrier. Experimental results show that while the TS stays at a roughly constant distance relative to the native state, it shifts with force relative to the unfolded state. Interestingly, a conversion of the protein extension into amino acid units shows that the TS position follows the Leffler–Hammond postulate: the higher the force, the lower the number of unzipped amino acids relative to the native state. The results are compared with the quasi-reversible unfolding–folding of a short DNA hairpin.

Excitonic coupling directly revealed on single chains of polyfluorene by combined force spectroscopy and fluorescence microscopy

Molecular aggregates were discovered in 1930’s, yet, the forces and excitonic coupling energy associated with the aggregate formation have not been detected so far. We directly measure such force and energy on single chains of the conjugated polymer polyfluorene using atomic force and fluorescence microscopes. The polyfluorene chain is attached on either side to a substrate and an AFM tip, respectively, and mechanically stretched under intense laser irradiation. The force – extension curves show force peaks that are attributed to gradual unfolding of the chain. Upon the irradiation, neighboring conjugated segments interact via excitonic coupling when in contact and experience an attractive force which is detected by the AFM. Analysis of the force curves provides excitonic coupling energy which is of same order as theoretically calculated values for a face-to-face fluorene dimer, and in agreement with the energy obtained from single-chain fluorescence spectra. Apart from contributing an essential piece of knowledge in the field of molecular photophysics, the work demonstrates on molecular scale a novel energy conversion mechanism from light to mechanical energy which could be potentially used, e.g., as a driving mechanism for molecular motors.

Microchemomechanical devices using DNA hybridization

The programmability of DNA oligonucleotides has led to sophisticated DNA nanotechnology and considerable research on DNA nanomachines powered by DNA hybridization. Here, we investigate an extension of this technology to the micrometer-colloidal scale, in which observations and measurements can be made in real time/space using optical microscopy and holographic optical tweezers. We use semirigid DNA origami structures, hinges with mechanical advantage, self-assembled into a nine-hinge, accordion-like chemomechanical device, with one end anchored to a substrate and a colloidal bead attached to the other end. Pulling the bead converts the mechanical energy into chemical energy stored by unzipping the DNA that bridges the hinge. Releasing the bead returns this energy in rapid (>20 μm/s) motion of the bead. Force-extension curves yield energy storage/retrieval in these devices that is very high. We also demonstrate remote activation and sensing—pulling the bead enables binding at a distant site. This work opens the door to easily designed and constructed micromechanical devices that bridge the molecular and colloidal/cellular scales.

Sugar-Pucker Force-Induced Transition in Single-Stranded DNA

The accurate knowledge of the elastic properties of single-stranded DNA (ssDNA) is key to characterize the thermodynamics of molecular reactions that are studied by force spectroscopy methods where DNA is mechanically unfolded. Examples range from DNA hybridization, DNA ligand binding, DNA unwinding by helicases, etc. To date, ssDNA elasticity has been studied with different methods in molecules of varying sequence and contour length. A dispersion of results has been reported and the value of the persistence length has been found to be larger for shorter ssDNA molecules. We carried out pulling experiments with optical tweezers to characterize the elastic response of ssDNA over three orders of magnitude in length (60–14 k bases). By fitting the force-extension curves (FECs) to the Worm-Like Chain model we confirmed the above trend:the persistence length nearly doubles for the shortest molecule (60 b) with respect to the longest one (14 kb). We demonstrate that the observed trend is due to the different force regimes fitted for long and short molecules, which translates into two distinct elastic regimes at low and high forces. We interpret this behavior in terms of a force-induced sugar pucker conformational transition (C3′-endo to C2′-endo) upon pulling ssDNA.

Pulling the springs of a cell by single-molecule force spectroscopy

The fundamental unit of the human body comprises of the cells which remain embedded in a fibrillar network of extracellular matrix proteins which in turn provides necessary anchorage the cells. Tissue repair, regeneration and reprogramming predominantly involve a traction force mediated signalling originating in the ECM and travelling deep into the cell including the nucleus via circuitry of spring-like filamentous proteins like microfilaments or actin, intermediate filaments and microtubules to elicit a response in the form of mechanical movement as well as biochemical changes. The ‘springiness’ of these proteins is highlighted in their extension–contraction behaviour which is manifested as an effect of differential traction force. Atomic force microscope (AFM) provides the magic eye to visualize and quantify such force-extension/indentation events in these filamentous proteins as well as in whole cells. In this review, we have presented a summary of the current understanding and advancement of such measurements by AFM based single-molecule force spectroscopy in the context of cytoskeletal and nucleoskeletal proteins which act in tandem to facilitate mechanotransduction.