Enhanced Emission Induced by FRET from a Long-Lifetime, Low Quantum Yield Donor to a Long-Wavelength, High Quantum Yield Acceptor

Abstract

We report observation of high quantum yield, long-lifetime fluorescence from a red dye BO-PRO-3 excited by resonance energy transfer (RET). The acceptor fluorescence was highly enhanced upon binding to the donor-labeled DNA. A ruthenium complex (Ru) was chosen as a donor in this system because of its long fluorescence lifetime. Both donor and acceptor were non-covalently bound to DNA. Emission from the donor-acceptor system (DA) at wavelengths exceeding 600 nm still preserves the long-lifetime component of the Ru donor, retaining average fluorescence lifetimes in the range of 30-50 ns. Despite the low quantum yield of the Ru donor in the absence of acceptor, its overall quantum yield of the DA pair was increased by energy transfer to the higher quantum yield acceptor BO-PRO-3. The wavelength-integrated intensity of donor and acceptor bound to DNA was many-fold greater than the intensity of the donor and acceptor separately bound to DNA. The origin of this effect is due to an efficient energy transfer from the donor, competing with non-radiative depopulation of the donor excited state. The distinctive features of DA complexes can be used in the development of a new class of engineered luminophores that display both long lifetime and long-wavelength emission. Similar DA complexes can be applied as proximity indicators, exhibiting strong fluorescence of adjacently located donors and acceptors over the relatively weak fluorescence of separated donors and acceptors.

Keywords: BO-PRO-3; DNA; Enhanced emission; fluorescence resonance energy transfer; metal-ligand complex.

Fig. 1.

Chemical structures of [Ru(bpy)₂(dppz)]²⁺ (Ru-BD) and BO-PRO-3.

Fig. 2.

Emission spectra of Ru-BD in the presence and absence of varying concentrations of the BO-PRO-3 acceptor bound to calf thymus DNA. The long dashed line shows the emission spectrum of the acceptor alone, 120 μM BO-PRO-3. The BO-PRO-3 concentrations are shown in μM.

Fig. 3.

Absorption (top) and uncorrected excitation (bottom) spectra of Ru-BD with 120 μM BO-PRO-3 donor-acceptor pair (short dashed lines) and the acceptor alone, 120 μM BO-PRO-3 (long dashed line) bound to calf thymus DNA. Ratios of two absorption (top) and two excitation (bottom) spectra are shown in solid lines.

Fig. 4.

Frequency-domain intensity decays of Ru-BD in the absence (top) and presence (bottom) of 30 μM BO-PRO-3 bound to calf thymus DNA. The middle panel presents the frequency responses of the BO-PRO-3 acceptor alone bound to DNA. The solid circles represent the phase or modulation values, and the solid lines show the best multi-exponential fits to the data.

Fig. 5.

Time-domain representation of intensity decays of Ru-BD bound to DNA in the absence and presence of 30 μM BO-PRO-3 bound to calf thymus DNA. The intensity decay of the BO-PRO-3 acceptor alone is also shown.

Scheme I.

A potential long-wavelength, long-lifetime luminophore based on a long-lifetime donor (D) and a short-lifetime acceptor (A).