High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics

Dulin, David; Cui, Tao; Cnossen, Jelmer; Docter, M.W.; Lipfert, Jan; Dekker, Nynke H.

doi:10.1016/j.bpj.2015.10.018

Cited by 84 publications

(103 citation statements)

References 70 publications

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“…Such scaling converts the raw SNR into the scaled SNR (sSNR), the SNR that would be observed for a 1 nm step at 1 Hz bandwidth (Moffitt, Chemla, Izhaky, & Bustamante, 2006). Using comparisons of sSNR from various experiments (Abbondanzieri, Greenleaf, Shaevitz, Landick, & Block, 2005; Derrington et al, 2015; Dulin et al, 2015; Holden et al, 2010) we generated Figure 9, which shows a log-log sensitivity plot, highlighting the large new phase-space available to SPRNT experiments. Specifically figure 9 was constructed as follows:

The diagonal line representing the spatiotemporal resolution limit for OT comes from (Moffitt et al, 2008).

…”

Section: Comparison To Other Single-molecule Techniquesmentioning

confidence: 99%

“…Spatiotemporal resolution of SPRNT (Derrington et al, 2015) as compared to OT (Abbondanzieri et al, 2005; Moffitt et al, 2006; Moffitt et al, 2008), MT (Dulin et al, 2015), and TIRF-FRET (Holden et al, 2010). Many nucleic acid processing enzymes work on time- scales and step sizes that are too fast and too small to be resolved with current techniques.…”

Section: Figurementioning

confidence: 99%

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Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT

Laszlo

Derrrington

Gundlach

2017

Methods in Enzymology

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Nanopores are emerging as new single-molecule tools in the study of enzymes. Based on progress in nanopore sequencing of DNA, a tool called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) was developed to measure the movement of enzymes along DNA in real time. In this new method, an enzyme is loaded onto a DNA (or RNA) molecule. A single stranded DNA end of this complex is drawn into a nanopore by an electrostatic potential that is applied across the pore. The single-stranded DNA passes through the pore’s constriction until the enzyme comes into contact with the pore. Further progression of the DNA through the pore is then controlled by the enzyme. An ion current that flows through the pore’s constriction is modulated by the DNA in the constriction. Analysis of ion current changes reveals the advance of the DNA with high spatiotemporal precision, thereby providing a real-time record of the enzyme’s activity. Using an engineered version of the protein nanopore MspA, SPRNT has spatial resolution as small as 40 pm at milliseconds time scales, while simultaneously providing the DNA’s sequence within the enzyme. In this chapter SPRNT is introduced and its extraordinary potential is exemplified using the helicase Hel308. Two distinct sub-states are observed for each one-nucleotide advance; one of these about half-nucleotide long steps is ATP-dependent and the other is ATP-independent. The spatiotemporal resolution of this low-cost single-molecule technique lifts the study of enzymes to a new level of precision, enabling exploration of hitherto unobservable enzyme dynamics in real-time.

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The diagonal line representing the spatiotemporal resolution limit for OT comes from (Moffitt et al, 2008).

…”

Section: Comparison To Other Single-molecule Techniquesmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT

Laszlo

Derrrington

Gundlach

2017

Methods in Enzymology

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“…This becomes especially important in studies of fast processes or those that involve small changes in DNA length. Such suppression is usually achieved by using higher forces, shorter DNAs and smaller beads [41,42]. All these factors increase the frequency of fluctuations (Equation 2) and necessitate the use of CMOS or high speed CCD cameras.…”

Section: Force Calibrationmentioning

confidence: 99%

“…Another example of measuring changes in DNA extension at single base pair resolution has been reported in a recent study by Dekker et al [42], who used a bright coherent laser source and a CMOS camera for fast image acquisition to resolve 3 Å steps with a 1 s period for immobile beads melted into the surface and 5 Å steps with a 0.5 s period for dsDNA anchored beads.…”

Section: Rna Polymerase Mediated Dna Scrunchingmentioning

confidence: 99%

A Guide to Magnetic Tweezers and Their Applications

Sarkar

Rybenkov

2016

Front. Phys.

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Magnetic force spectroscopy is a rapidly developing single molecule technique that has found numerous applications at the interface of physics and biology. Since the invention of the first magnetic tweezers, a number of modifications to the approach have helped to relieve the limitations of the original design while amplifying its strengths. Inventive molecular biology solutions further advanced the technique by expanding its possible applications. In its present form, the method can be applied to both single molecules and live cells without resorting to intense irradiation, can be easily multiplexed, accommodates multiple DNAs, displays impressive resolution, and allows a remarkable ease in the stretching and twisting of macromolecules. In this review, we describe the architecture of magnetic tweezers, key requirements for experimental design and analysis of data, and outline several applications of the method that illustrate its versatility.

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“…Although most traditional MT systems are unable to achieve comparable spatial resolution to OT experiments, more recent advances in MT techniques have also demonstrated the capability of monitoring nanometer scale displacements [20,21]. In either case, to study the mechanical properties of a system of interest (ie.…”

Section: Introductionmentioning

confidence: 99%

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid

Long

Parks

Stone

2016

Methods

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Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules.

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High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics

Cited by 84 publications

References 70 publications

Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT

Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT

A Guide to Magnetic Tweezers and Their Applications

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid

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