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

Cherny, Dmitry; Eperon, Ian C.; Bagshaw, Clive R.

doi:10.1007/s00249-008-0383-z

Cited by 23 publications

(18 citation statements)

References 55 publications

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“…The FRET efficiency ( E ) can be obtained based on the change of donor emission in the presence ( I DA ) and absence ( I D ) of the acceptor 1 (2B). 52−55 …”

Section: Resultsmentioning

confidence: 99%

Dual-Channel Single-Molecule Fluorescence Resonance Energy Transfer to Establish Distance Parameters for RNA Nanoparticles

Shu

Zhang

Petrenko³

et al. 2010

ACS Nano

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The increasing interest in RNA nanotechnology and the demonstrated feasibility of using RNA nanoparticles as therapeutics have prompted the need for imaging systems with nanometer-scale resolution for RNA studies. Phi29 dimeric pRNAs can serve as building blocks in assembly into the hexameric ring of the nanomotors, as modules of RNA nanoparciles, and as vehicles for specific delivery of therapeutics to cancers or viral infected cells. The understanding of the 3D structure of this novel RNA dimeric particle is fundamentally and practically important. Although a 3D model of pRNA dimer has been proposed based on biochemical analysis, no distance measurements or X-ray diffraction data have been reported. Here we evaluated the application of our customized single-molecule dual-viewing system for distance measurement within pRNA dimers using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Ten pRNA monomers labeled with single donor or acceptor fluorophores at various locations were constructed and eight dimers were assembled. smFRET signals were detected for six dimers. The tethered arm sizes of the fluorophores were estimated empirically from dual-labeled RNA/DNA standards. The distances between donor and acceptor were calculated and used as distance parameters to assess and refine the previously reported 3D model of the pRNA dimer. Distances between nucleotides in pRNA dimers were found to be different from those of the dimers bound to procapsid, suggesting a conformational change of the pRNA dimer upon binding to the procapsid.

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“…The FRET efficiency ( E ) can be obtained based on the change of donor emission in the presence ( I DA ) and absence ( I D ) of the acceptor 1 (2B). 52−55 …”

Section: Resultsmentioning

confidence: 99%

Dual-Channel Single-Molecule Fluorescence Resonance Energy Transfer to Establish Distance Parameters for RNA Nanoparticles

Shu

Zhang

Petrenko³

et al. 2010

ACS Nano

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“…Recently, RNA cubic scaffolds 71 were constructed using several RNA sequences that do not fold on themselves but self-assemble with one another in a defined manner. This strategy is reminiscent of DNA nanotechnology, but in contrast to DNA strategies, RNA synthesis can be coupled to RNA self-assembly to generate fully assembled RNA cubes during in vitro transcription.…”

Section: Techniques For Constructing Rna Nanoparticlesmentioning

confidence: 99%

The emerging field of RNA nanotechnology

2010

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RNA can be designed and manipulated just like DNA while having different rules for base-pairing and displaying functions similar to proteins. The large variety of loops and motifs in RNA allow them to fold into numerous complicated structures. This diversity provides a platform for identifying viable building blocks for particle assemblies, substrate binding and manufacture engineering. RNA thermal stability allows production of multivalent nanostructures with defined stoichiometry. Here we review the unique qualities of RNA nanotechnology and their distinct properties inside the body. We describe techniques for constructing RNA nanoparticles from different building blocks and their applications in nanomedicine. Finally, we discuss challenges in predicting and synthesizing RNA and offer some perspectives on the yield and cost of RNA production.

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“…This shot noise leads to histograms of FRET efficiency that are distributed in an approximately Gaussian manner and with a predictable width (Gopich and Szabo, 2005; Nir et al, 2006; Cherny et al, 2009; Chung et al, 2009; Kalinin et al, 2010) (Figure 2). However, conformational dynamics of the molecule(s) can lead to temporal variations in the distance between a donor/acceptor pair and correspondingly dynamic variations in FRET efficiency levels.…”

Section: Conversion Of Fret Efficiency To Distancementioning

confidence: 99%

Three-dimensional molecular modeling with single molecule FRET

Brunger

Strop

Vrljic

et al. 2011

Journal of Structural Biology

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Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.

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

Cited by 23 publications

References 55 publications

Dual-Channel Single-Molecule Fluorescence Resonance Energy Transfer to Establish Distance Parameters for RNA Nanoparticles

Dual-Channel Single-Molecule Fluorescence Resonance Energy Transfer to Establish Distance Parameters for RNA Nanoparticles

The emerging field of RNA nanotechnology

Three-dimensional molecular modeling with single molecule FRET

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