MAGNETIC TWEEZERS
Introduction
Force spectroscopy methods are a group of single-molecule biophysical techniques that are used to investigate physical and mechanical properties of biological macromolecules or polymers by measuring their behavior under mechanical force. The main force spectroscopy techniques used include atomic force microscopy, optical tweezers, acoustic force microscopy, and magnetic force spectroscopy.
Magnetic force spectroscopy (MFS) is performed using magnetic tweezers apparatus in which macromolecules are attached to paramagnetic microbeads that are then manipulated using a controllable external magnetic field. The position of each magnetic particle is determined using a microscopic objective and a camera, allowing the response of the macromolecule to the applied force to be measured.
It is a versatile technique that has provided insights across biophysical and biological applications and is gaining increasing interest, particularly as technological progress leads to improvements in magnetic tweezer performance.
Principles & history
Force spectroscopy was pioneered by the invention in 1986 of optical tweezers, or optical traps, which manipulate objects as small as a single atom using a focused light beam. From this concept, in the early 1990s, magnetic tweezers were developed to allow the manipulation of paramagnetic beads which were attached to macromolecules.1-3 Both initial studies focused on the mechanical properties of single molecules of DNA. For example, Croquette’s group at Ecole Normale Supérieure (ENS),1 attached a double-stranded DNA molecule at one end to a glass surface and at the other end to a paramagnetic microbead. They then manipulated the bead using a pair of magnets and the response of the tethered molecule to the applied force was monitored by tracking the bead position. The study showed sharp transitions in the elasticity of DNA related to its overwound or underwound states which had significant implications for understanding the behavior of DNA mechanics under conditions such as during transcription or replication.
In the basic magnetic tweezer set up, the experiment is performed in a glass flow cell, which is on top of an inverted microscope with permanent magnets above it. The macromolecule being studied is then tethered between the flow cell surface and a paramagnetic bead. The force applied to the bead can be varied by moving the magnets with a motorized drive, exerting differing degrees of force on the tethered molecules. A range of imaging methods are used to track the bead position, with accurate tracking along the vertical axis often achieved from diffraction patterns of beads placed slightly out of focus by design. An example set up is shown in the figure below – taken from.4
Figure from Eeftens et al 2015.4 a) Schematic of an example magnetic tweezers set-up for measuring on a tethered DNA molecule. An LED illuminates the flow cell through a lens and the magnet holder. Imaging is performed with a 50x objective onto a camera. Magnets manipulate a magnetic bead attached to the DNA. b) A flow cell is constructed with coverslips. The bottom coverslip is amine-coated and has reference beads bound to it. The top coverslip has holes to allow fluid flow. c) Schematic of a tethered DNA molecule. A biotinylated DNA molecule is linked to a streptavidin-coated paramagnetic bead, and to azide groups on the flow cell surface via DBCO at the other end. CC BY 4.0
Applications – biophysics, biology
Magnetic tweezers are powerful and versatile instruments, that are compatible with a wide range of experimental set ups and are therefore well suited for diverse applications across biophysics and biology. They have brought insights to many biomolecular systems (reviewed in 5-7) including:
- The mechanical properties of macromolecules including DNA, RNA and double stranded RNA
- The actions of nucleic acid-binding enzymes such as helicases, DNA and RNA polymerases, topoisomerases, and restriction enzymes
- The effect of small molecule ligands binding to RNA structures.8
- Nucleoprotein filaments, protein-mediated DNA condensation, and nucleosome stability (reviewed by 5)
- The mechanical properties of living cells 9,10 and the viscoelastic properties of the cytoplasm 11
Furthermore, because magnetic tweezers allow a constant force to be applied over a long period of time, including at low forces (<1 pN), they have been successfully used to study the dynamics of protein folding and unfolding (reviewed in 5) and protein-protein interactions.12
Advantages, limitations & advances
Whilst historically a significant drawback of magnetic tweezers was low temporal and spatial resolution due to the data acquisition via video-microscopy, recent advances in tracking algorithms, illumination, and imaging strategies have brought magnetic tweezers on a par with optical tweezers in terms of spatiotemporal resolution.5,6 Another significant advance has been multiplexing, with some magnetic tweezer systems allowing simultaneous analysis of tens or even hundreds of molecules in parallel.13
However, although magnetic tweezers are a well-established apparatus, there has been no widely available commercial instrument until now, and magnetic force spectroscopy has only been accessible to specialized laboratories.
Depixus MAGNA One™
Depixus™ has now made magnetic force spectroscopy available to the wider research community with the commercial launch of the MAGNA One instrument, cartridge and reagent system. MAGNA One provides high-resolution analysis of individual biomolecular interactions across thousands of molecules simultaneously, unlocking a wealth of kinetic, structural, and functional information.
By bringing high scaling to a single-molecule technique, MAGNA One reveals biological variability in stunning detail. Rare, but biologically relevant, conformations or binding events, and heterogeneity can be detected providing a deeper understanding of molecular behavior. MAGNA One is therefore set to transform the landscape of biophysics research and improve the hit-to-lead and lead optimization stages of drug development.
References
1. Strick TR, Allemand JF, Bensimon D, Bensimon A, Croquette V. The elasticity of a single supercoiled DNA molecule. Science. 1996;271(5257):1835-1837. doi:10.1126/science.271.5257.1835
2. Smith SB, Cui Y, Bustamante C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science. 1996;271(5250):795-799. doi:10.1126/science.271.5250.795
3. Smith SB, Finzi L, Bustamante C. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science. 1992;258(5085):1122 LP – 1126. doi:10.1126/science.1439819
4. Eeftens JM, van der Torre J, Burnham DR, Dekker C. Copper-free click chemistry for attachment of biomolecules in magnetic tweezers. BMC Biophys. 2015;8:9. doi:10.1186/s13628-015-0023-9
5. Dulin D. An Introduction to Magnetic Tweezers. Methods in Molecular Biology. 2024;2694:375-401. doi: 10.1007/978-1-0716-3377-9_18
6. Choi HK, Kim HG, Shon MJ, Yoon TY. High-Resolution Single-Molecule Magnetic Tweezers. Annu Rev Biochem. 2022;91:33-59. doi:10.1146/annurev-biochem-032620-104637
7. Sarkar, R., & Rybenkov V V. A guide to magnetic tweezers and their applications. Front Phys. 2016;4:48. doi:10.3389/fphy.2016.00048
8. Parmar S, Bume DD, Connelly CM, et al. Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression. Nat Commun. 2024;15(1):8173. doi:10.1038/s41467-024-52235-3
9. Bonakdar N, Schilling A, Spörrer M, et al. Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes. Exp Cell Res. 2015;331(2):331-337. doi:10.1016/j.yexcr.2014.10.001
10. Aermes C, Hayn A, Fischer T, Mierke CT. Environmentally controlled magnetic nano-tweezer for living cells and extracellular matrices. Sci Rep. 2020;10(1):13453. doi:10.1038/s41598-020-70428-w
11. Bausch AR, Möller W, Sackmann E. Measurement of local viscoelasticity and forces in living cells by magnetic tweezers. Biophys J. 1999;76(1 Pt 1):573-579. doi:10.1016/S0006-3495(99)77225-5
12. Bauer MS, Gruber S, Hausch A, et al. Single-molecule force stability of the SARS-CoV-2-ACE2 interface in variants-of-concern. Nat Nanotechnol. 2024;19(3):399-405. doi:10.1038/s41565-023-01536-7
13. De Vlaminck I, Henighan T, van Loenhout MTJ, et al. Highly parallel magnetic tweezers by targeted DNA tethering. Nano Lett. 2011;11(12):5489-5493. doi:10.1021/nl203299e