Fluorescence resonance energy transfer (FRET) and competing processes in donor–acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

Dietrich, Anja; Buschmann, Volker; Müller, Christian; Sauer, Markus

doi:10.1016/s1389-0352(01)00039-3

Cited by 140 publications

(179 citation statements)

References 72 publications

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“…The FRET D-lifetime measurements, presented in this study, were performed for optimal labeling conditions (ideally each D has an A). Given this, we were able to distinguish also low FRET efficiencies from each other ( 19 , we did not observe dimerization (no shift in the absorption and emission spectra of the fluorophores bound to dsDNA). However, in the FCS measurements, we did observe a strongly reduced molecular brightness of A for the 10 ∆bp sample in comparison to the 15 ∆bp sample.…”

Section: Fret Efficiencies Determined By D-lifetime Measurementsmentioning

confidence: 71%

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

et al. 2011

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Inter-or intramolecular distances of biomolecules can be studied by Förster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleedthrough, thereby tolerating a high excess (up to 100 fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50 % in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in-vitro and in-vivo FRET-based spectroscopy and imaging.

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Section: Fret Efficiencies Determined By D-lifetime Measurementsmentioning

confidence: 71%

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

et al. 2011

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“…Although the fluorescent probes are anchored to the bases by a linker with a flexible component, the model-derived and experimental distances are in remarkable agreement (17,18,44).…”

Section: Structural Support For the Initially-bound Complex Modelmentioning

confidence: 85%

“…Coupling of fluorescein-5-isothiocyanate (FITC) with these primary amines to form a thiourea linkage then provides FRET donors at various positions, for measurement of a progression of donor-acceptor distances. The strands are designed such that the nearest guanine base is at least 6 base pairs away from the TAMRA label to avoid quenching of TAMRA by guanine (17). All constructs are fully duplex and have the same DNA sequence and length.…”

Section: Distance Measurements By Dna-to-dna Fretmentioning

confidence: 99%

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

et al. 2007

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T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein.Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the Cterminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. FRET measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogs incorporated at various positions in the DNA we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7-8 bases and is sufficient to allow synthesis of a 3 base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.

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“…A sensitive tool for monitoring the assembly of macromolecules and their kinetics is fluorescence microscopy, by virtue of imaging the dynamics of fluorescent dyes attached to the molecules of interest. Recently, fluorescent imaging techniques have been developed for individual molecules, allowing the monitoring of molecular interactions at the single molecule level (Blanchard et al 2004;Coban et al 2006;Deniz et al 1999;Dietrich et al 2002;Friedman et al 2006;Gell et al 2006;Ha 2001Ha , 2004Merchant et al 2007;Murphy et al 2004;Myong et al 2006;Rueda et al 2004;Yasuda et al 2003). These new techniques provide unique information about the natural heterogeneity (structural and dynamics) of individual macromolecules, and allow the analysis of interactions between the molecules, the dynamics of complex formation, determination of the stoichiometry of the complex and calculation of the spatial distance between specific molecules in a complex.…”

Section: Introductionmentioning

confidence: 99%

“…In many cases, the information about inter-dye distance is extracted from FRET efficiency histograms which are often approximated by a Gaussian function (Agrawal et al 2008;Coban et al 2006;Deniz et al 1999;Dietrich et al 2002;Ha 2001;Iqbal et al 2008a; Kapanidis et al 2004;Koopmans et al 2007; Kuzmenkina et al 2006;Lee et al 2005;Merchant et al 2007;Murphy et al 2004;Nir et al 2006;Sabanayagam et al 2005;Schuler et al 2005;Sugawa et al 2007;Yildiz and Selvin 2005;Yim et al 2005;Zhang et al 2007). The apparent width of thus extracted histograms is usually in the range of 0.07-0.2 (on a scale of 0-1) and often ascribed to variations of the interdye distance (Coban et al 2006;Dietrich et al 2002;Merchant et al 2007;Schuler et al 2005;Sugawa et al 2007). Here we describe an approach to discern the contributions of various factors to the apparent width of FRET distribution, showing that photon shot noise and modulations of orientation factor due to helix flexibilities are the main sources for the experimentally observed width of the FRET distribution.…”

Section: Introductionmentioning

confidence: 99%

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

2008

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Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1).Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.

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Fluorescence resonance energy transfer (FRET) and competing processes in donor–acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

Cited by 140 publications

References 72 publications

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

Förster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

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