Correction-free force calibration for magnetic tweezers experiments

Ostrofet, Eugeniu; Papini, Flávia S.; Dulin, David

doi:10.1038/s41598-018-34360-4

Cited by 39 publications

(46 citation statements)

References 59 publications

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“…High throughput magnetic tweezers apparatus. The high throughput magnetic tweezers apparatus has been previously described (15,18,35). Shortly, it is a custom inverted microscope with a 50× oil immersion objective (CFI Plan Achro 50 XH, NA 0.9, Nikon, Germany), on top of which a flow cell is mounted.…”

Section: Methodsmentioning

confidence: 99%

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Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

Seifert

Nies

Papini

et al. 2020

Preprint

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AbstractRNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

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Section: Methodsmentioning

confidence: 99%

“…Flow cell fabrication. The flow cell assembly has been described previously (35). The flow cell was mounted on the magnetic tweezers setup and rinsed with 1 ml of 1×phosphate buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

Seifert

Nies

Papini

et al. 2020

Preprint

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“…The evident discrepancy between the model prediction and the estimations might be due to an inhomogeneity in the magnitude of force applied to the magnetic beads since the amount of embedded iron oxide particles is not stringently constant. 64 This could explain the non-monotonic variation of the pause probability around 5 pN OF. Alternatively, the sharp increase in the off-pathway pause probability total at large OFs might indicate an additional force dependency, e.g., in the entry rate to the elemental pause; this is compatible with the recent observation that the elemental pause may be in fact a half-translocated state.…”

Section: All Transcriptional Pauses Compete With the Active Catalyticmentioning

confidence: 98%

Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

Janissen

Eslami-Mossallam

Artsimovitch

et al. 2019

Preprint

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Pausing by RNA polymerase (RNAp) facilitates the recruitment of regulatory factors, RNA folding, and other related processes. While backtracking and intra-structural isomerization have been proposed to trigger pausing, the mechanisms of pause formation and recovery remain debated. Using high-throughput magnetic tweezers with single-molecule sensitivity, we have examined the full temporal spectrum of Escherichia coli RNAp transcription dynamics. Together with probabilistic dwell-time analysis and modelling, this has identified three distinct states that compete with elongation: a short-lived elemental pause and two longlived backtracked pause states. We demonstrate that recovery from backtracking is not governed by diffusional Brownian motion, but rather by intrinsic RNA cleavage, whereby RNAp conformational changes hinder cleavage in long-lived pause states. We further show that state switching underlies stochastic alterations in the frequency of short pauses. Based on these findings, we propose a consensus model of intrinsic pausing that unifies all key findings while resolving earlier contradictions.

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“…To control the quality of the nucleic acid scaffolds, we used the high throughput magnetic tweezers apparatus previously described in Ref. (29,31,32). A typical field of view (FoV) is shown in Supplementary Figure 2.…”

Section: High Throughput Magnetic Tweezersmentioning

confidence: 99%

“…We also provide new protocols to generate similar RNA scaffolds. Using high throughput magnetic tweezers (29,31,32), we evaluate quantitatively the quality and the purity of the scaffolds, and we show that our new strategy enables the fabrication of DNA hairpins in fewer steps and with a higher purity and yield than the existing methods (21,22). We also provide new protocols to synthesize long stem RNA hairpin, i.e.…”

Section: Introductionmentioning

confidence: 99%

High-yield fabrication of DNA and RNA scaffolds for single molecule force and torque spectroscopy experiments

Papini

Seifert

Dulin

2019

Preprint

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Single molecule biophysics experiments have enabled the observation of biomoleculeswith a great deal of precision in space and time, e.g. nucleic acids mechanical properties and protein-nucleic acids interactions using force and torque spectroscopy techniques.The success of these experiments strongly depends on the capacity of the researcher to design and fabricate complex nucleic acid scaffolds, as the pertinence and the yield of the experiment strongly depend on the high quality and purity of the final scaffold.Though the molecular biology techniques involved are well known, the fabrication of nucleic acids scaffold for single molecule experiments still remains a difficult task. Here, we present new protocols to generate high quality coilable double-stranded DNA and RNA, as well as DNA and RNA hairpins with ~500-1000 bp long stems. Importantly, we present a new approach based on single-stranded DNA's annealing and show, using magnetic tweezers, that it is more efficient to generate complex nucleic acid scaffolds in larger amount and at higher purity than a standard PCR-digestion-ligation approach. The protocols we describe here enable the design of any sort of complex nucleic acid scaffold for single molecule biophysics experiments and will therefore be extremely valuable to the community.

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Correction-free force calibration for magnetic tweezers experiments

Cited by 39 publications

References 59 publications

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

High-yield fabrication of DNA and RNA scaffolds for single molecule force and torque spectroscopy experiments

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