The Structure of Cyanine 5 Terminally Attached to Double-Stranded DNA: Implications for FRET Studies

Iqbal, Asif; Wang, Lihua; Thompson, Katherine C.; Lilley, David M. J.; Norman, D.

doi:10.1021/bi800773f

Cited by 98 publications

(105 citation statements)

References 35 publications

(49 reference statements)

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“…Therefore, we took advantage of the FRET effect of the common Cy3/Cy5 fluorophore pair in single-molecule fluorescence studies, which involves nucleic acids. 32 FRET is a powerful tool for retrieving information on molecular proximity, orientation, and confirmation on the nm scale from samples. 33 By applying double-labeled oligonucleotides with Cy3 as the donor on the 3′-end and Cy5 as the acceptor on the 5′-end of the oligonucleotide sequence, first experiments with pure oligonucleotides, that means without encapsulation and without the presence of cells, were performed via fluorescent measurements ( Figure S9).…”

Section: Fret Measurementsmentioning

confidence: 99%

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells

Tomcin¹,

Baier²,

Landfester³

et al. 2014

IJN

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For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly( n -butylcyanoacrylate) (PBCA) nanocapsules as a shell and separately an oligonucleotide (20 mer) as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides) reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.

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Section: Fret Measurementsmentioning

confidence: 99%

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells

Tomcin¹,

Baier²,

Landfester³

et al. 2014

IJN

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“…1 It has been widely used for measuring the distance between two fluorophores. [2][3][4] However, as a spectroscopic ruler conventional FRET suffers from a limited length scale of approximately 10 nm. Plasmonic structures provide a route towards longer range non-radiative energy transfer (NRET) via nanometal surface energy transfer (NSET) or localised surface plasmon (LSP) coupled FRET.…”

Section: Introductionmentioning

confidence: 99%

Experimental and Theoretical Investigation of the Distance Dependence of Localized Surface Plasmon Coupled Förster Resonance Energy Transfer

et al. 2014

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The distance dependence of localized surface plasmon (LSP) coupled Förster resonance energy transfer (FRET) is experimentally and theoretically investigated using a trilayer structure composed of separated monolayers of donor and acceptor quantum dots with an intermediate Au nanoparticle layer. The dependence of the energy transfer efficiency, rate, and characteristic distance, as well as the enhancement of the acceptor emission, on the separations between the three constituent layers is examined. A d –4 dependence of the energy transfer rate is observed for LSP-coupled FRET between the donor and acceptor planes with the increased energy transfer range described by an enhanced Förster radius. The conventional FRET rate also follows a d –4 dependence in this geometry. The conditions under which this distance dependence is valid for LSP-coupled FRET are theoretically investigated. The influence of the placement of the intermediate Au NP is investigated, and it is shown that donor–plasmon coupling has a greater influence on the characteristic energy transfer range in this LSP-coupled FRET system. The LSP-enhanced Förster radius is dependent on the Au nanoparticle concentration. The potential to tune the characteristic energy transfer distance has implications for applications in nanophotonic devices or sensors.

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“…Our earlier NMR studies have shown that when these are attached to the 5Ј termini of duplex DNA via a 3-carbon linker to the 5Ј-phosphate they are predominantly stacked onto the ends of the helix in the manner of an additional base pair (11,26). This would provide a favorable situation in which the orientation dependence of FRET could be observed, and we have therefore studied a series of DNA and DNA-RNA hybrid duplexes to seek this effect.…”

mentioning

confidence: 99%

“…Although this is a good approximation for freely mobile fluorophores like fluorescein that are terminally attached to double-stranded nucleic acids, it is rather less probable for cyanine dyes in this situation. By using NMR we have shown that Cy3 and Cy5 are predominantly stacked onto the end of dsDNA when coupled to the 5Ј-terminal phosphates via C 3 linkers (11,26) (Fig. 1 C and D).…”

mentioning

confidence: 99%

“…The transition moments for the to * transitions lie in the plane of the indole rings close to the polymethyne linker (26), and are therefore directed in planes that are approximately parallel to each other. This situation approximates to ⍜ D ϭ ⍜ A ϭ 90°, and where ⍜ T depends on the length and geometry of the DNA helix and the angles between the terminal base pairs and the transition moments.…”

mentioning

confidence: 99%

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Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

Iqbal

Arslan

Okumuş

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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318

369

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We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5 ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Fö rster transfer obeys the orientation dependence as expected for a dipole-dipole interaction.cyanine fluorophores ͉ FRET ͉ kappa squared ͉ single-molecule FRET F luorescence resonance energy transfer (FRET) has become widely used to report on distances over the macromolecular scale in biology (1), reviewed in refs. 2-4. The method is highly sensitive, and consequently has been widely exploited in singlemolecule experiments in biological systems. Energy transfer results from dipolar coupling between the transition moments of two fluorophores, and the efficiency of the process (E FRET ) depends on the separation between the donor and acceptor fluorophores, raised to the sixth power. Although such data are frequently interpreted on the assumption of a simple relationship between E FRET and distance, E FRET should also depend on the relative orientation of the transition dipole vectors.The orientation dependence is likely to be most significant where the fluorophores are constrained (5-9). This has been demonstrated experimentally by using a fluorophore that was terminally affixed to duplex DNA by two points of covalent attachment (10), thereby seriously constraining its motion. This situation is not typical of most FRET studies involving nucleic acids. Fluorophores are normally tethered by a single point of attachment, and in theory would be significantly less constrained. But if the fluorophores adopt a rigid manner of attachment to the helix, an orientational dependence could be observed.Cy3 and Cy5 are a commonly used fluorophore pair, especially in single-molecule experiments. Our earlier NMR studies have shown that when these are attached to the 5Ј termini of duplex DNA via a 3-carbon linker to the 5Ј-phosphate they are predominantly stacked onto the ends of the helix in the manner of an additio...

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The Structure of Cyanine 5 Terminally Attached to Double-Stranded DNA: Implications for FRET Studies

Cited by 98 publications

References 35 publications

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells

Experimental and Theoretical Investigation of the Distance Dependence of Localized Surface Plasmon Coupled Förster Resonance Energy Transfer

Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

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