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

Abstract

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...

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

“…1). If not otherwise stated, we assume that both dyes stack to the end base pairs of the DNA/RNA helix, with their planes perpendicular to the helix axis (Iqbal et al 2008a, b;Norman et al 2000).…”

“…L 0 should be considered as the sum of the Cy3 and Cy5 rises (8 Å ; Iqbal et al 2008a) L D and L A can be considered as off-centre distances for donor and acceptor, respectively. Variation for r due to helix rotation leads to r r ¼ L D L A r j sin ujr tot : Assuming that L D and L A are less than 10 Å each, whereas r is *46 or 50 Å (14 9 2.7 Å ?…”

“…Recently, it has been shown that Cy3 and Cy5 fluorophores are fixed via stacking to the terminal base pairs of DNA/RNA duplex with their transition vectors perpendicular to the helix axis (Iqbal et al 2008a, b), forming a static angle u relative to each other. For Helix bending and twisting can be simulated by rotating the vector f A,0 around the axes z and y by angle a and s, corresponding to the helix bending and twisting, respectively (note that the scheme shown on Fig.…”

“…An analysis of Cy3 conjugated to a DNA duplex has suggested the presence of a species with an unstacked fluorophore either in *12% (Sanborn et al 2007) or *17% (Iqbal et al 2008a) of molecules. With a DNA/RNA duplex the fraction of unstacked Cy3 is less (*6% Iqbal et al 2008a).…”

“…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).…”