Biology subjects

Wong, W. P.

Publications and source records attributed to Wong, W. P..

7 recordsLinked to original sources

bioRxiv2025

Light-guided molecular patterning for programmable multiplexed single-molecule manipulation

Single-molecule force spectroscopy enables the detailed probing of molecular interactions, providing new insights into molecular mechanisms--yet studying biology "one molecule at a time" can lead to throughput challenges that limit applications. While multiplexed single-molecule assays can address these issues, suitable functionalization of surfaces is required, which remains a technical challenge--many commonly used approaches are constrained by random and sparse biomolecules arrangements, limiting programmability and throughput. An ideal anchoring method would enable (i) high surface densities to maximize throughput, (ii) precise control of spatial position and molecular identity for maximum control, (iii) covalent linking for high force application, and (iv) efficient patterning without the need for expensive facilities to maximize accessibility. To achieve these aims, we have developed a light-guided surface patterning method that can covalently organize oligonucleotides (oligos) without the need for lithographic equipment. Oligos with 3-Cyanovinylcarbazole (CNVK) nucleoside are crosslinked by UV patterns reflected through a digital micromirror device (DMD), with beads arranged accordingly. To demonstrate compatibility with established single-molecule methods, we performed single-molecule force spectroscopy experiments on patterned coverslips, using both magnetic tweezers and hydrodynamic-based systems. Our light-guided approach provides a scalable and accessible solution for biomolecular patterning that allows precise control over molecular identity and spatial positioning, enabling high-throughput measurements in single-molecule research.

biophysics↗

bioRxiv2025

High-Throughput Centrifuge Force Microscopy Reveals Dynamic Immune-Cell Avidity at the Single-Cell Level

Cell-cell binding, mediated by the physical interactions of receptors and their ligands, plays a fundamental role in immune processes such as immune surveillance and T-cell activation. However, current approaches for measuring cell avidity often lack either throughput or quantitative precision. Here, we introduce a high-throughput approach for quantifying cell binding lifetimes and strength using a centrifuge force microscope (CFM)--a compact microscope operating within a standard benchtop centrifuge. The CFM enables live monitoring of single-cell interactions under force, conducting thousands of force experiments in parallel. To facilitate the real-time study of live cell interactions, we developed a next-generation CFM with multichannel fluorescence imaging capabilities. This system accommodates measurements in two modes: cell-protein binding and cell-cell avidity assays. Using this system, we investigated immune-cell binding mediated by Bispecific T-cell Engager (BiTE) molecules, a novel immunotherapy designed to enhance immune-cell targeting of cancer cells. In cell-protein assays, we quantified T- and B-cell unbinding from BiTE-functionalized surfaces, revealing receptor-specific relationships between ligand concentration and binding strength. In cell-cell assays, we examined BiTE-mediated binding of T-cells to Nalm6 B-cells, a precursor leukemia cell line, uncovering a strong, time-dependent increase in BiTE-mediated immune-cell avidity. By integrating high-throughput and quantitative single-cell force analysis, the CFM provides new insights into the dynamic nature of immunological interactions under force, with broad implications for immunotherapy and cellular mechanics.

biophysics↗

bioRxiv2023

Mapping Single-molecule Protein Complexes in 3D with DNA Nanoswitch Calipers

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force-spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices we call DNA Nanoswitch Calipers, which we have previously introduced, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5[A] agreement to previously reported structures.

biophysics↗

bioRxiv2020

DNA nanoswitch barcodes for multiplexed biomarker profiling

The detection of molecular biomarkers plays a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. The ability to detect multiple and diverse molecular biomarkers within a single accessible assay would have great utility, providing a more comprehensive picture for clinical evaluation and research, but is a challenge with standard methods. One promising approach is the use of dynamic DNA nanostructures that can respond to molecular biomarkers, which have recently been used in a variety of biosensing strategies. In this work, we report the use of programmable DNA nanoswitches for the multiplexed detection of up to 6 biomarkers within a single pot through the use of a barcoded gel-based readout. We demonstrate the barcoding capability using gene fragments that correspond to 6 different diseases, with each fragment or combination of fragments producing a unique barcode signature. As a defining feature of our method, we show "mixed multiplexing" for simultaneous barcoded detection of different types of biomolecules - DNA, RNA, antibody and protein in a single assay. To demonstrate clinical potential, we show multiplexed detection of a prostate cancer biomarker panel in serum that includes two microRNA sequences and prostate specific antigen (PSA). This strategy holds promise in clinical diagnostics for profiling complex and diverse biomarker panels.

bioengineering↗

bioRxiv2020

Dual functions of Aire CARD multimerization in the transcriptional regulation of T cell tolerance

Aggregate-like biomolecular assemblies are emerging as new conformational states with functionality. Aire, a transcription factor essential for central T cell tolerance, is known to form large aggregate-like assemblies visualized as nuclear foci. We demonstrate that Aire utilizes Caspase Activation Recruitment Domain (CARD) to form filamentous homo-multimers in vitro, and this assembly mediates foci formation and transcriptional activity. However, CARD-mediated multimerization is a double-edged sword as it also makes Aire susceptible to interaction with PML bodies, sites of many nuclear processes including protein quality control of nuclear aggregates. Several loss-of-function Aire mutants, including those causing autoimmune polyendocrine syndrome type-1, form foci with increased PML body association. Directing Aire to PML bodies impairs Aires transcriptional activity, while dispersing PML bodies with a viral antagonist restores it. Thus, our study reveals a new regulatory role of PML bodies in Aire function and highlights the interplay between nuclear aggregate-like assemblies and PML-mediated quality control.

immunology↗

bioRxiv2019

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

Single-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors, to the mechanical response of soft materials within the cell. To expand the single-molecule toolbox, we have developed a surface-free force spectroscopy assay based on a high-speed hydrodynamic trap capable of applying extremely high tensions for long periods of time. High-speed single-molecule trapping is enabled by a rigid and gas-impermeable microfluidic chip, rapidly and inexpensively fabricated out of glass, double-sided tape and UV-curable adhesive. Our approach does not require difficult covalent attachment chemistries, and enables simultaneous force application and single-molecule fluorescence. Using this approach, we have induced a highly extended state with twice the contour length of B-DNA in regions of partially intercalated double-stranded (dsDNA) by applying forces up to 250 pN. This highly extended state resembles the hyperstretched state of dsDNA, which was initially discovered as a structure fully intercalated by dyes under high tension. It has been hypothesized that hyperstretched DNA could also be induced without the aid of intercalators if high-enough forces were applied, which matches our observation. Combining force application with single-molecule fluorescence imaging is critical for distinguishing hyperstretched DNA from single-stranded DNA that can result from peeling. High-speed hydrodynamic trapping is a powerful yet accessible force spectroscopy method that enables the mechanics of biomolecules to be probed in previously difficult to access regimes.

biophysics↗

bioRxiv2019

The Dynamic Strength of the Hair-Cell Tip Link Reveals Mechanisms of Hearing and Deafness

Our senses of hearing and balance rely on the extraordinarily sensitive molecular machinery of the inner ear to convert deflections as small as the width of a single carbon atom1,2 into electrical signals that the brain can process3. In humans and other vertebrates, transduction is mediated by hair cells4, where tension on tip links conveys force to mechanosensitive ion channels5. Each tip link comprises two helical filaments of atypical cadherins bound at their N-termini through two unique adhesion bonds6-8. Tip links must be strong enough to maintain a connection to the mechanotransduction channel under the dynamic forces exerted by sound or head movement--yet might also act as mechanical circuit breakers, releasing under extreme conditions to preserve the delicate structures within the hair cell. Previous studies have argued that this connection is exceptionally static, disrupted only by harsh chemical conditions or loud sound9-12. However, no direct mechanical measurements of the full tip-link connection have been performed. Here we describe the dynamics of the tip-link connection at single-molecule resolution and show how avidity conferred by its double stranded architecture enhances mechanical strength and lifetime, yet still enables it to act as a dynamic mechanical circuit breaker. We also show how the dynamic strength of the connection is facilitated by strong cis-dimerization and tuned by extracellular Ca2+, and we describe the unexpected etiology of a hereditary human deafness mutation. Remarkably, the connection is several thousand times more dynamic than previously thought, challenging current assumptions about tip-link stability and turnover rate, and providing insight into how the mechanotransduction apparatus conveys mechanical information. Our results reveal fundamental mechanisms that underlie mechanoelectric transduction in the inner ear, and provide a foundation for studying multi-component linkages in other biological systems.

biophysics↗