Raman Spectroscopy of Optically Trapped Living Human T Cell Subsets and Monocytes.

In recent years, Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past, the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study, we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally, we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications.

Spatiotemporal formation of a single liquid-like condensate and amyloid fibrils of α-synuclein by optical trapping at solution surface.

Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to ß-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Quantification of the interaction forces between dengue virus and dopamine type-2 receptor using optical tweezers.

BACKGROUND: Dengue virus (DENV) causes the most significant mosquito-borne viral disease with a wide spectrum of clinical manifestation, including neurological symptoms associated with lethal dengue diseases. Dopamine receptors are expressed in central nervous system, and dopamine antagonists have been reported to exhibit antiviral activity against DENV infection in vivo and in vitro. Although identification of host-cell receptor is critical to understand dengue neuropathogenesis and neurotropism, the involvement of dopamine receptors in DENV infection remains unclear. RESULTS: We exploited the sensitivity and precision of force spectroscopy to address whether dopamine type-2 receptors (D2R) directly interact with DENV particles at the first step of infection. Using optical tweezers, we quantified and characterized DENV binding to D2R expressed on Chinese hamster ovary (CHO) cells. Our finding suggested that the binding was D2R- and DENV-dependent, and that the binding force was in the range of 50-60 pN. We showed that dopamine antagonists prochlorperazine (PCZ) and trifluoperazine (TFP), previously reported to inhibit dengue infection, interrupt the DENV-D2R specific binding. CONCLUSIONS: This study demonstrates that D2R could specifically recognize DENV particles and function as an attachment factor on cell surfaces for DENV. We propose D2R as a host receptor for DENV and as a potential therapeutic target for anti-DENV drugs.

Nanomechanics of wild-type and mutant dimers of the inner-ear tip-link protein protocadherin 15.

Mechanical force controls the opening and closing of mechanosensitive ion channels atop the hair bundles of the inner ear. The filamentous tip link connecting transduction channels to the tallest neighboring stereocilium modulates the force transmitted to the channels and thus changes their probability of opening. Each tip link comprises four molecules: a dimer of protocadherin 15 (PCDH15) and a dimer of cadherin 23, all of which are stabilized by Ca2+ binding. Using a high-speed optical trap to examine dimeric PCDH15, we find that the protein's mechanical properties are sensitive to Ca2+ and that the molecule exhibits limited unfolding at a physiological Ca2+ concentration. PCDH15 can therefore modulate its stiffness without undergoing large unfolding events under physiological conditions. The experimentally determined stiffness of PCDH15 accords with published values for the stiffness of the gating spring, the mechanical element that controls the opening of mechanotransduction channels. When PCDH15 exhibits a point mutation, V507D, associated with nonsyndromic hearing loss, unfolding events occur more frequently under tension and refolding events occur less often than for the wild-type protein. Our results suggest that the maintenance of appropriate tension in the gating spring is critical to the appropriate transmission of force to transduction channels, and hence to hearing.

Single-molecule force spectroscopy of toehold-mediated strand displacement.

Toehold-mediated strand displacement (TMSD) is extensively utilized in dynamic DNA nanotechnology and for a wide range of DNA or RNA-based reaction circuits. Investigation of TMSD kinetics typically relies on bulk fluorescence measurements providing effective, bulk-averaged reaction rates. Information on individual molecules or even base pairs is scarce. In this work, we explore the dynamics of strand displacement processes at the single-molecule level using single-molecule force spectroscopy with a microfluidics-enhanced optical trap supported by state-of-the-art coarse-grained simulations. By applying force, we can trigger and observe TMSD in real-time with microsecond and nanometer resolution. We find TMSD proceeds very rapidly under load with single step times of 1 µs. Tuning invasion efficiency by introducing mismatches allows studying thousands of forward/backward invasion events on a single molecule and analyze the kinetics of the invasion process. Extrapolation to zero force reveals single step times for DNA invading DNA four times faster than for RNA invading RNA. We also study the kinetics of DNA invading RNA, a process that in the absence of force would rarely occur. Our results reveal the importance of sequence effects for the TMSD process and have relevance for a wide range of applications in nucleic acid nanotechnology and synthetic biology.

Acousto-dielectric tweezers enable independent manipulation of multiple particles.

Acoustic tweezers have gained substantial interest in biology, engineering, and materials science for their label-free, precise, contactless, and programmable manipulation of small objects. However, acoustic tweezers cannot independently manipulate multiple microparticles simultaneously. This study introduces acousto-dielectric tweezers capable of independently manipulating multiple microparticles and precise control over intercellular distances and cyclical cell pairing and separation for detailed cell-cell interaction analysis. Our acousto-dielectric tweezers leverage the competition between acoustic radiation forces, generated by standing surface acoustic waves (SAWs), and dielectrophoretic (DEP) forces, induced by gradient electric fields. Modulating these fields allows for the precise positioning of individual microparticles at points where acoustic radiation and DEP forces are in equilibrium. This mechanism enables the simultaneous movement of multiple microparticles along specified paths as well as cyclical cell pairing and separation. We anticipate our acousto-dielectric tweezers to have enormous potential in colloidal assembly, cell-cell interaction studies, disease diagnostics, and tissue engineering.

High Stability and Low Power Nanometric Bio-Objects Trapping through Dielectric-Plasmonic Hybrid Nanobowtie.

Micro and nano-scale manipulation of living matter is crucial in biomedical applications for diagnostics and pharmaceuticals, facilitating disease study, drug assessment, and biomarker identification. Despite advancements, trapping biological nanoparticles remains challenging. Nanotweezer-based strategies, including dielectric and plasmonic configurations, show promise due to their efficiency and stability, minimizing damage without direct contact. Our study uniquely proposes an inverted hybrid dielectric-plasmonic nanobowtie designed to overcome the primary limitations of existing dielectric-plasmonic systems, such as high costs and manufacturing complexity. This novel configuration offers significant advantages for the stable and long-term trapping of biological objects, including strong energy confinement with reduced thermal effects. The metal's efficient light reflection capability results in a significant increase in energy field confinement (EC) within the trapping site, achieving an enhancement of over 90% compared to the value obtained with the dielectric nanobowtie. Numerical simulations confirm the successful trapping of 100 nm viruses, demonstrating a trapping stability greater than 10 and a stiffness of 2.203 fN/nm. This configuration ensures optical forces of approximately 2.96 fN with an input power density of 10 mW/µm2 while preserving the temperature, chemical-biological properties, and shape of the biological sample.

High-Performance Suspension Bead Sensor Based on Optical Tweezers and Immuno-Rolling Circle Amplification.

In recent years, optical tweezers have become an effective bioassay tool due to their unique advantages, especially in combination with suspension beads, which can be applied to develop a high-performance analysis platform capable of high-quality imaging and stable signal output. However, the optical tweezer-assisted bead analysis is still at the early stage, and further development of different favorable methods is in need. Herein, we have first developed the optical tweezer-assisted immuno-rolling circle amplification (immuno-RCA) on beads for protein detection. Prostate-specific antigen was selected as the model analyte, and the immunosandwich structure on beads was built by the high affinity of "antibody-antigen". The "protein-nucleic acid" signals were effectively converted through the covalent coupling procedure of antibodies and oligonucleotides, further initiating the RCA reaction to achieve signal amplification. The individual beads with the strong irregular Brownian motion in a fluid environment were eventually trapped by the optical tweezers to acquire the accurate and high-quality signal. Compared with the conventional immunoassay on beads, the sensitivity of the developed strategy was increased by 587 times with a limit of detection of 4.29 pg/mL (0.13 pM), as well as excellent specificity, stability, and reproducibility. This study developed the new optical tweezer-assisted beads imaging strategy for protein targets, which has great potential for being applied to clinical serology research and expands the application of optical tweezers in the bioassays.

Parsing digital or analog TCR performance through piconewton forces.

αß T cell receptors (TCRs) principally recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP366-374/Db and PA224-233/Db, respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker up-regulation in vitro. Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies.

Tunneling nanotubes mediate KRas transport: Inducing tumor heterogeneity and altering cellular membrane mechanical properties.

Mutation in oncogene KRas plays a crucial role in the occurrence and progression of numerous malignant tumors. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. We hypothesize that oncogene KRas mutations are intrinsic to alterations in cellular mechanics that promote malignant tumor generation and progression. Here, we demonstrate the use of optical tweezers coupled with a confocal fluorescence imaging system and gene interference technique to reveal that the mutant KRas protein can be transported between homogeneous and heterogeneous tumor cells by tunneling nanotubes (TNTs), resulting in a significant reduction of membrane tension and acceleration of membrane phospholipid flow in the recipient cells. Simultaneously, the changes in membrane mechanical properties of the tumor cells also enhance the metastatic and invasive ability of the tumors, which further contribute to the deterioration of the tumors. This finding helps to clarify the association between oncogene mutations and changes in the mechanical properties of tumor cells, which provides a theoretical basis for the development of cancer treatment strategies. STATEMENT OF SIGNIFICANCE: Here, we present a laser confocal fluorescence system integrated with optical tweezers to observe the transfer of mutant KRasG12D protein from mutant cells to wild-type cells through TNTs. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. Our results demonstrate a significant decrease in membrane tension and an increase in membrane phospholipid flow in recipient cells. These alterations in mechanical properties augment the migration and invasive capabilities of tumor cells, contributing to tumor malignancy. Our findings propose that cellular mechanical properties could serve as new markers for tumor development, and targeting membrane tension may hold potential as a therapeutic strategy.

Measurements of Spontaneous and External Stimuli Molecular Release Processes from a Single Optically Trapped Poly(lactic-co-glycolic) Acid Microparticle and a Liposome Containing Gold Nanospheres.

We investigated the single particle kinetics of the molecular release processes from two types of microcapsules used as drug delivery systems (DDS): biodegradable poly(lactic-co-glycolic) acid (PLGA) and a light-triggered-degradable liposome encapsulating gold nanospheres (liposome-GNP). To optimize the design of DDS capsules, it is highly desirable to develop a method for real-time monitoring of the release process. Using a combination of optical tweezers and confocal fluorescence microspectroscopy we successfully analyzed a single optically trapped PLGA particle and liposome-GNPs in solution. From temporal decay profiles of the fluorescence intensity, we determined the time constant τ of the release processes. We demonstrated that the release rate of spontaneously degradable microcapsules (PLGA) decreased with increasing size, while conversely, the release rate of external stimuli-degradable microcapsules (liposome-GNPs) increased in proportion to their size. This result is explained by the differences in the disruption mechanisms of the capsules, with PLGA undergoing hydrolysis and the GNPs in the liposome-GNP undergoing a photoacoustic effect under nanosecond pulsed laser irradiation. The present approach offers a way forward to an alternative microanalysis system for single drug delivery nanocarriers.

Kinesin-1-transported liposomes prefer to go straight in 3D microtubule intersections by a mechanism shared by other molecular motors.

Kinesin-1 ensembles maneuver vesicular cargoes through the three-dimensional (3D) intracellular microtubule (MT) network. To define how such cargoes navigate MT intersections, we first determined how many kinesins from an ensemble on a lipid-based cargo simultaneously engage a MT, and then determined the directional outcomes (straight, turn, terminate) for liposome cargoes at perpendicular MT intersections. Run lengths of 350-nm diameter liposomes decorated with up to 20, constitutively active, truncated kinesin-1 KIF5B (K543) were longer than single motor transported cargo, suggesting multiple motor engagement. However, detachment forces of lipid-coated beads with ~20 kinesins, measured using an optical trap, showed no more than three simultaneously engaged motors, with a single engaged kinesin predominating, indicating anticooperative MT binding. At two-dimensional (2D) and 3D in vitro MT intersections, liposomes frequently paused (~2 s), suggesting kinesins simultaneously bind both MTs and engage in a tug-of-war. Liposomes showed no directional outcome bias in 2D (1.1 straight:turn ratio) but preferentially went straight (1.8 straight:turn ratio) in 3D intersections. To explain these data, we developed a mathematical model of liposome transport incorporating the known mechanochemistry of kinesins, which diffuse on the liposome surface, and have stiff tails in both compression and extension that impact how motors engage the intersecting MTs. Our model predicts the ~3 engaged motor limit observed in the optical trap and the bias toward going straight in 3D intersections. The striking similarity of these results to our previous study of liposome transport by myosin Va suggests a "universal" mechanism by which cargoes navigate 3D intersections.

Optical torque calculations and measurements for DNA torsional studies.

The angular optical trap (AOT) is a powerful instrument for measuring the torsional and rotational properties of a biological molecule. Thus far, AOT studies of DNA torsional mechanics have been carried out using a high numerical aperture oil-immersion objective, which permits strong trapping but inevitably introduces spherical aberrations due to the glass-aqueous interface. However, the impact of these aberrations on torque measurements is not fully understood experimentally, partly due to a lack of theoretical guidance. Here, we present a numerical platform based on the finite element method to calculate forces and torques on a trapped quartz cylinder. We have also developed a new experimental method to accurately determine the shift in the trapping position due to the spherical aberrations by using a DNA molecule as a distance ruler. We found that the calculated and measured focal shift ratios are in good agreement. We further determined how the angular trap stiffness depends on the trap height and the cylinder displacement from the trap center and found full agreement between predictions and measurements. As a further verification of the methodology, we showed that DNA torsional properties, which are intrinsic to DNA, could be determined robustly under different trap heights and cylinder displacements. Thus, this work has laid both a theoretical and experimental framework that can be readily extended to investigate the trapping forces and torques exerted on particles with arbitrary shapes and optical properties.

Quantitative Determination of DNA Bridging Efficiency of Chromatin Proteins.

DNA looping is important for genome organization in all domains of life. The basis of DNA loop formation is the bridging of two separate DNA double helices. Detecting DNA bridge formation generally involves the use of complex single-molecule techniques (atomic force microscopy, magnetic or optical tweezers). Although DNA bridging can be qualitatively described, quantification of DNA bridging and bridging dynamics using these techniques is challenging. Here we describe a biochemical assay capable of not only detecting DNA bridge formation but also allowing for quantification of DNA bridging efficiency and the quantification of the effects of physicochemical conditions or protein interaction partners on DNA bridge formation.

Unravelling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers.

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers, in particular, have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA-organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to a magnetic bead inside a flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleoprotein filaments can be extracted from the data.

Nanopore tweezers show fractional-nucleotide translocation in sequence-dependent pausing by RNA polymerase.

RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein-DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.

Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants.

Many neurodegenerative diseases feature misfolded proteins that propagate via templated conversion of natively folded molecules. However, crucial questions about how such prion-like conversion occurs and what drives it remain unsolved, partly because technical challenges have prevented direct observation of conversion for any protein. We observed prion-like conversion in single molecules of superoxide dismutase-1 (SOD1), whose misfolding is linked to amyotrophic lateral sclerosis. Tethering pathogenic misfolded SOD1 mutants to wild-type molecules held in optical tweezers, we found that the mutants vastly increased misfolding of the wild-type molecule, inducing multiple misfolded isoforms. Crucially, the pattern of misfolding was the same in the mutant and converted wild-type domains and varied when the misfolded mutant was changed, reflecting the templating effect expected for prion-like conversion. Ensemble measurements showed decreased enzymatic activity in tethered heterodimers as conversion progressed, mirroring the single-molecule results. Antibodies sensitive to disease-specific epitopes bound to the converted protein, implying that conversion produced disease-relevant misfolded conformers.

Robust, high-yield, rapid fabrication of DNA constructs for Magnetic Tweezers.

Single-molecule techniques are highly sensitive tools that can reveal reaction intermediates often obscured in experiments involving large ensembles of molecules. Therefore, they provide unprecedented information on the mechanisms that control biomolecular reactions. Currently, one of the most significant single-molecule assays is Magnetic Tweezers (MT), which probes enzymatic reactions at high spatio-temporal resolutions on tens, if not hundreds, of molecules simultaneously. For high-resolution MT experiments, a short double-stranded DNA molecule (less than 2000 base pairs) is typically attached between a micron-sized superparamagnetic bead and a surface. The fabrication of such a substrate is key for successful single-molecule assays, and several papers have discussed the possibility of improving the fabrication of short DNA constructs. However, reported yields are usually low and require additional time-consuming purification steps (e.g., gel purification). In this paper, we propose the use of a Golden Gate Assembly assay that allows for the production of DNA constructs within minutes (starting from PCR products). We discuss how relevant parameters may affect the yield and offer single-molecule experimentalists a simple yet robust approach to fabricate DNA constructs.

TMPyP binding evokes a complex, tunable nanomechanical response in DNA.

TMPyP is a porphyrin capable of DNA binding and used in photodynamic therapy and G-quadruplex stabilization. Despite its broad applications, TMPyP's effect on DNA nanomechanics is unknown. Here we investigated, by manipulating λ-phage DNA with optical tweezers combined with microfluidics in equilibrium and perturbation kinetic experiments, how TMPyP influences DNA nanomechanics across wide ranges of TMPyP concentration (5-5120 nM), mechanical force (0-100 pN), NaCl concentration (0.01-1 M) and pulling rate (0.2-20 µm/s). Complex responses were recorded, for the analysis of which we introduced a simple mathematical model. TMPyP binding, which is a highly dynamic process, leads to dsDNA lengthening and softening. dsDNA stability increased at low (<10 nM) TMPyP concentrations, then decreased progressively upon increasing TMPyP concentration. Overstretch cooperativity decreased, due most likely to mechanical roadblocks of ssDNA-bound TMPyP. TMPyP binding increased ssDNA's contour length. The addition of NaCl at high (1 M) concentration competed with the TMPyP-evoked nanomechanical changes. Because the largest amplitude of the changes is induced by the pharmacologically relevant TMPyP concentration range, this porphyrin derivative may be used to tune DNA's structure and properties, hence control the wide array of biomolecular DNA-dependent processes including replication, transcription, condensation and repair.