Studying DNA–protein interactions with single-molecule Förster resonance energy transfer

Farooq, Shazia; Fijen, Carel; Hohlbein, Johannes

doi:10.1007/s00709-013-0596-6

Cited by 17 publications

(17 citation statements)

References 113 publications

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“…A classic example for such a molecular ruler is Förster-type resonance energy transfer (FRET) 5 , which allows achieving structural information with a spatial resolution in the nanometre range and (sub)millisecond temporal resolution 6 7 8 9 10 . However, other photophysical effects such as photo-induced electron transfer (PET) 11 12 13 14 or protein-induced fluorescence enhancement (PIFE) 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 can be used for similar purposes.…”

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confidence: 99%

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Ploetz

Lerner

Husada

et al. 2016

Sci Rep

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104

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Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.

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mentioning

confidence: 99%

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Ploetz

Lerner

Husada

et al. 2016

Sci Rep

Self Cite

104

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“…Information obtained from these experiments is crucially important for building mechanistic models of diverse reactions. 1,2 Nano-or micro-scopic platforms combined with microscopy techniques become very popular and allow accessing information that is otherwise hidden. [3][4][5] However, most of the SM techniques cannot be parallelized and are often technically challenging.…”

Section: Introductionmentioning

confidence: 99%

Oriented Soft DNA Curtains for Single Molecule Imaging

Kopūstas

Ivanovaite

Rakickas

et al. 2020

Preprint

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Over the past twenty years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flowstretch assay of stably immobilized and oriented DNA molecules using protein template-directed assembly. In our assay a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended either by single-or both-ends immobilization to the protein templates. For oriented both-end DNA immobilization we employed heterologous DNA labeling and protein template coverage with the anti-digoxigenin antibody. In contrast to the single-end, both-ends immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g. CRISPR-Cas9) binding assay that monitors binding location and position of individual fluorescently labeled proteins on DNA.

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“…10 In the last two decades, techniques with single-molecule sensitivity and resolution allowed to overcome temporal and spatial averaging inherent to ensemble based characterisations. 11,12 For DNA polymerases, different experimental designs have been applied ranging from optical or magnetic traps 13 (reviewed by Heller et al 14 ) to conductivity measurements on protein nanopores 15,16 and applications utilising Förster resonance energy transfer (FRET), a process in which the distance-dependent energy transfer from a donor fluorophore to an acceptor chromophore allows to resolve changes of distances in the nanometre range. 17,18 Single-molecule FRET experiments on DNA polymerases have been designed to measure the rate of DNA polymerization by stranddisplacing DNA polymerases, 19 to identify sliding characteristics and binding orientations of HIV reverse transcriptase, 20,21 to identify translocation of DNA polymerases with single base pair resolution 22 and to determine conformational dynamics and the free-energy landscapes of pre-chemistry nucleotide selection in E.coli DNA polymerase I.…”

Section: Introductionmentioning

confidence: 99%

A single-molecule FRET sensor for monitoring DNA synthesis in real time

Fijen

Silva

Hochkoeppler

et al. 2017

Phys. Chem. Chem. Phys.

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We developed a versatile DNA assay and framework for monitoring polymerization of DNA in real time and at the single-molecule level. The assay consists of an acceptor labelled DNA primer annealed to a DNA template that is labelled on its single stranded, downstream overhang with a donor fluorophore. Upon extension of the primer using a DNA polymerase, the overhang of the template alters its conformation from a random coil to the canonical structure of double stranded DNA. This conformational change increases the distance between the donor and the acceptor fluorophore and can be detected as a decrease in the Förster resonance energy transfer (FRET) efficiency between both fluorophores. Remarkably, the DNA assay does not require any modification of the DNA polymerase and albeit the simple and robust spectroscopic readout facilitates measurements even with conventional fluorimeters or stopped-flow equipment, single-molecule FRET provides additional access to parameters such as the processivity of DNA synthesis and, for one of the three DNA polymerases tested, the detection of binding and dissociation of the DNA polymerase to DNA. We furthermore demonstrate that primer extensions by a single base can be resolved.

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Studying DNA–protein interactions with single-molecule Förster resonance energy transfer

Cited by 17 publications

References 113 publications

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Oriented Soft DNA Curtains for Single Molecule Imaging

A single-molecule FRET sensor for monitoring DNA synthesis in real time

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