Measuring Two at the Same Time: Combining Magnetic Tweezers with Single-Molecule FRET

Abstract

Molecular machines are the workhorses of the cell that efficiently convert chemical energy into mechanical motion through conformational changes. They can be considered powerful machines, exerting forces and torque on the molecular level of several piconewtons and piconewton-nanometer, respectively. For studying translocation and conformational changes of these machines, fluorescence methods, like FRET, as well as "mechanical" methods, like optical and magnetic tweezers, have proven well suited over the past decades. One of the current challenges in the field of molecular machines is gaining maximal information from single-molecule experiments by simultaneously measuring translocation, conformational changes, and forces exerted by these machines. In this chapter, we describe the combination of magnetic tweezers with single-molecule FRET for orthogonal simultaneous readout to maximize the information gained in single-molecule experiments.

Investigating supramolecular systems using Förster resonance energy transfer

“…Besides the development of FRET signal observation techniques, the current trend toward integrating FRET with other techniques will likely continue. 158,159 For example, much effort is currently focused on developing super-resolution FRET to allow the phenomena monitored through FRET to be localized with unprecedented spatial resolution. 160 In addition, FRET-AFM has shown great potential in simultaneously monitoring and manipulating molecules, 33,161 and FRET-mass spectrometry techniques are revealing molecules’ gas-phase behavior.…”

High throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

“…Specialized single molecule force measurements (such as atomic force microscopy, optical traps and magnetic tweezers) have been advancing rapidly over the past two decades and have revealed how force is used in biological systems, from individual proteins to complexes and from individual organelles to cells (Elosegui-Artola et al, 2017, Seo et al, 2016, Yao et al, 2016, Lherbette et al, 2017. More recently, these techniques have been combined with highresolution single molecule fluorescence approaches and cell imaging to visualise dynamic systems under the controlled application of force (Cordova et al, 2014, Madariaga-Marcos et al, 2018, Newton et al, 2019, Swoboda et al, 2014. These measurements have started to probe cellular systems in order to dissect mechanotransduction pathways.…”

Combined Magnetic Tweezers and Micro-mirror Total Internal Reflection Fluorescence Microscope for Single-Molecule Manipulation and Visualization

“…Questions concerning the number of enzymes associated with activity, their binding affinity, their direction of movement on the DNA, and the relationship between binding and mechanical activity, among many others are challenging, if not impossible, to resolve solely with manipulation-based measurements. Recently, a hybrid technique [21–24] that combines MT with total internal reflection fluorescent microscopy (TIRF) was developed, which overcomes these limitations on understanding DNA-enzyme interactions by providing orthogonal information to the mechanical signal obtained with conventional magnetic tweezers.…”