Over the past two decades, we have witnessed remarkable advances in the scale and resolution of genomics, transcriptomics, and proteomics. These technologies have generated powerful new insights into the underpinnings of health and disease.
But to truly grasp the mechanisms of life, we must look even deeper than today’s mainstream technologies allow – exploring the interactions of individual molecules that drive cellular function and behavior.
Many to one: from bulk to single molecule analysis
Life depends on interactions between biological molecules – DNA, RNA, proteins, lipids and more.
These molecular interactions vary in strength, duration, and frequency, introducing a rich layer of complexity within cells that cannot be resolved either through single-cell measurements or averaged molecular data.
Even seemingly identical proteins or nucleic acids can differ in chemical modifications or conformational states, dramatically altering their function. Weak, transient, or rare interactions, which are easily masked in bulk analyses, can have outsized biological importance.
Characterizing individual biomolecular interactions in detail is therefore one of the keys to unravelling the complexities of life and developing more effective therapeutics. Yet achieving this in practice is far from simple.
Techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) can deliver results at scale by averaging data from millions of molecules. However, these bulk measurements obscure molecular heterogeneity and subtle binding behaviors that may be biologically or therapeutically significant.
Single-molecule techniques, such as optical tweezers, offer far greater resolution by enabling direct observation of individual molecular events. However, their low throughput, high technical demands, and reliance on specialized equipment make them impractical for widespread use, particularly in industrial settings.
To fully unlock the potential of single-molecule analysis, researchers must overcome the dual challenges of resolution and scalability.
One to many: the challenges of scaling magnetic force spectroscopy
Magnetic force spectroscopy (MFS), also known as magnetic tweezers, is a powerful single-molecule technique first developed in the 1990s.
Individual biomolecules such as DNA or RNA are attached at one end to a flow cell surface and to magnetic micron-scale beads at the other, which are then manipulated by a controllable external magnetic field. A camera system tracks the movements of the beads using interferometry, enabling precise measurement of the forces applied and the corresponding molecular response.
Despite its power and versatility, the widespread adoption of MFS has been hindered by its technical complexity and inherent lack of scalability. While early attempts achieved parallel tracking of up to 34 beads (1), several technical barriers have prevented larger-scale deployment. For example:
Conventional MFS systems rely on interferometric images for the optical detection of bead position, requiring thousands of camera pixels to detect a single bead and limiting the number of beads that can be detected in parallel.
MFS systems struggle when closely packed or irregularly spaced beads interfere with each other’s movement or when their diffraction patterns overlap, making bead tracking unreliable or impossible.
Cameras must combine large fields of view with high frame rates and pixel densities to detect small, fast displacements – a demanding and expensive specification.
However, a number of recent technological innovations in the field of MFS are overcoming these limitations, delivering significant improvements in accuracy and scale.
Magnetic force spectroscopy at scale
Recent advances have partially addressed the throughput of MFS, allowing tracking of hundreds or even thousands of beads in parallel (2-4) along with improvements in temporal resolution via higher frequency data acquisition (5,6).
However, the use of interferometric images to measure bead position is still a limitation in the scalability of MFS. For example, the tracking system in the recently developed stereo dark-field interferometry magnetic tweezers uses approximately 64x64 camera pixels per bead (2).
Alternative approaches based on Total Internal Reflection (TIR) illumination (7-10) – where the processing of an interferometric image is replaced by a simpler measurement of the brightness of a bead – are now being combined with highly sensitive cameras and machine-vision lensing to enable one-to-one bead-to-pixel mapping.
Another innovation lies in sample loading. Multi-use cartridges printed with a regular array of tens of thousands of microscopic spots maintain sufficient separation between individual molecules and their associated beads, preventing physical and optical interference. This efficient placement also avoids wasted space, enabling sample density to be increased by around 10-fold without compromising precision (11).
Together, these innovations enable high-resolution measurement of tens of thousands of molecular interactions in parallel – a scale previously unimaginable for single-molecule methods.
For the first time, these capabilities are being combined in user-friendly laboratory instruments, such as Depixus’ MAGNA One system – ranked second in The Analytical Scientist’s Innovation Awards 2024 – making MFS a scalable and accessible technology that can be used by researchers across academia and industry.
These tools enable researchers to explore the frontiers of molecular biology, teasing out the complexities of biology as it really happens. Novel applications are emerging for tackling challenging drug targets such as RNA, and innovative nucleic acid scaffolds are being developed to investigate protein-protein interactions, develop molecular glues, and characterize protein variants.
By combining single-molecule precision (many-to-one) with high-throughput analysis (one-to-many), the latest MFS platforms open up new possibilities for advancing biology and medicine.
Just as advances in sequencing technologies ushered in the era of single-cell biology, today’s breakthroughs in MFS are making large-scale single-molecule analysis a reality and setting the stage for a breakthrough in our understanding of life at the molecular level.
Newsletters
Receive the latest analytical science news, personalities, education, and career development – weekly to your inbox.
References
- N Ribeck, OA Saleh, “Multiplexed single-molecule measurements with magnetic tweezers,” Rev Sci Instrum, 79, 94301 (2008). DOI: 10.1063/1.2981687.
- M Rieu et al., “Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry,” Sci Adv, 7, (2021). DOI: 10.1126/sciadv.abe3902.
- R Agarwal, KE Duderstadt, “Multiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision,” Nat Commun, 11, 4714 (2020). DOI: 10.1038/s41467-020-18456-y.
- KC Johnson et al., “A multiplexed magnetic tweezer with precision particle tracking and bi-directional force control,” J Biol Eng, 11, 47 (2017). DOI: 10.1186/s13036-017-0091-2.
- D Dulin et al., “High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics,” Biophys J, 109, 2113 (2015). DOI: 10.1016/j.bpj.2015.10.018.
- A Huhle et al., “Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy,” Nat Commun, 6, 5885 (2015). DOI: 10.1038/ncomms6885.
- DC Prieve, “Measurement of colloidal forces with TIRM,” Adv Coll Int Sci, 82, 93 (1999). DOI: 10.1016/S0001-8686(99)00012-3.
- M Singh-Zocchi et al., “Single-molecule detection of DNA hybridization,” PNAS, 100, 7605 (2003). DOI: 10.1073/pnas.1337215100.
- P Lebel et al., “Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension,” Nat Methods, 11, 456 (2014). DOI: 10.1038/nmeth.2854.
- PM Oliver, JS Park, D Vezenov, “Quantitative high-resolution sensing of DNA hybridization using magnetic tweezers with evanescent illumination,” Nanoscale, 3, 581 (2011). DOI: 10.1039/c0nr00479k.
- I De Vlaminck, C Dekker, “Recent advances in magnetic tweezers,” Annu Rev Biophys, 41, 453 (2012). DOI: 10.1146/annurev-biophys-122311-100544.