Self-assembled donor comprising quantum dots and fluorescent proteins for long-range fluorescence resonance energy transfer.
We report on the development of a self-assembled donor for long-range fluorescence resonance energy transfer (FRET). To this end, a three-chromophore FRET (3Ch-FRET) system was constructed, which consists of a luminescent quantum dot (QD), enhanced yellow fluorescent proteins (EYFP), and Atto647-dye-modified oligonucleotides. The system was assembled by electrostatic binding of covalent EYFP-ssDNA conjugate to the QD and subsequent hybridization with complementary oligonucleotides labeled with Atto647-dye. The final conjugates comprise three different two-chromophore FRET (2Ch-FRET) subsystems, QD/EYFP, QD/Atto647, and EYFP/Atto647, respectively, which were studied in detail by steady-state and time-resolved photoluminescence measurements. The helicity of DNA allowed us to control donor/acceptor separations and thus enabled the detailed analysis of the various FRET processes. We found that the 2Ch-FRET and the 3Ch-FRET (QD/EYFP/Atto647) systems revealed FRET efficiencies and transfer rates that were affected by the availability of distinct FRET pathways. The derived energy-transfer efficiencies and Förster radii indicated that within the 3Ch-FRET system, the 2Ch-FRET subsystem QD/EYFP showed highest FRET efficiencies ranging from 64 to 72%. Thus, it can be used as a powerful donor system that combines the intrinsic advantages of QDs (large and spectrally broad absorption cross section) and EYFP (high quantum yield) and enables long-distance FRET processes for donor-acceptor distances of up to 13 nm.
Lu
Huachang
H
Technische Universität Dortmund, Fakultät Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund, Germany.
Schöps
Oliver
O
Woggon
Ulrike
U
Niemeyer
Christof M
CM
D001426:Bacterial Proteins / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D045663:Quantum Dots; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D008164:Luminescent Proteins; D009841:Oligonucleotides; C054562:yellow fluorescent protein, Bacteria