Extending Förster resonance energy transfer measurements beyond 100 Å using common organic fluorophores: enhanced transfer in the presence of multiple acceptors

Abstract

Abstract. Using commercially available organic fluorophores, the current applications of Förster (fluorescence) resonance energy transfer (FRET) are limited to about 80 Å. However, many essential activities in cells are spatially and/or temporally dependent on the assembly/disassembly of transient complexes consisting of large-size macromolecules that are frequently separated by distances greater than 100 Å. Expanding the accessible range for FRET to 150 Å would open up many cellular interactions to fluorescence and fluorescence-lifetime imaging. Here, we demonstrate that the use of multiple randomly distributed acceptors on proteins/antibodies, rather than the use of a single localized acceptor, makes it possible to significantly enhance FRET and detect interactions between the donor fluorophore and the acceptor-labeled protein at distances greater than 100 Å. A simple theoretical model for spherical bodies that have been randomly labeled with acceptors has been developed. To test the theoretical predictions of this system, we carried out FRET studies using a 30-mer oligonucleotide-avidin system that was labeled with the acceptors DyLight649 or Dylight750. The opposite 5′-end of the oligonucleotide was labeled with the Alexa568 donor. We observed significantly enhanced energy transfer due to presence of multiple acceptors on the avidin protein. The results and simulation indicate that use of a nanosized body that has been randomly labeled with multiple acceptors allows FRET measurements to be extended to over 150 Å when using commercially available probes and established protein-labeling protocols.

FRET from single to multiplexed signaling events

“…For n identical equidistant acceptors with a single donor, R 0 6 effectively multiplies by n , leading to a change of (1/ n ) r 6 in the distance term. As a consequence, the distance sensitivity of FRET increases with the presence of multiple acceptors per donor without a real change in molecular distance (Fábián et al 2010; Maliwal et al 2012; Walczewska-Szewc et al 2013 (Figs. 3 and 4a).…”

“…An extension of the multiplexing designs for intramolecular reporters involves consecutive ‘three-fluorophore’ or ‘two-step’ FRET (Maliwal et al 2012; Watrob et al 2003) (Fig. 1b).…”

Extending the range of FRET—the Monte Carlo study of the antenna effect

“…Figure 2 shows the dependence of the FRET efficiency in the presence of 1, 5 and 10 acceptors attached to the spherical protein with 40 Å diameter, as a function of the distance R . R 0 was assumed to be 80 Å, accordingly to recently available fluorescence probes [5, 24] and the distance is approximated by the rigid linker. Two orientation cases, random (black lines) and parallel (light blue lines), were considered.…”

“…The volume of attached protein remains the same in all cases and equals to volume enclosed by a sphere with radius r  = 20 Å. This volume is regarded to be adequate to the volume of typical miniantibody [5].

Fig.

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“…In order to test our model and show the influence of using different distance distributions on the final results, we used experimental results described by Maliwal et al [5] As the experimental antenna system they used the 30-mer ds oligonucleotide with biotin on the 3′-end and Alexa-568 (donor, A568) on the 5′-end. Earlier studies have shown that the distance between opposite ends of the DNA fragment varies around 100 Å in the absence of conformational changes [27].…”