Department of Biotechnology, University of Turku, FI-20520 Turku, Finland.
J Phys Chem B. 2011 Nov 24;115(46):13685-94. doi: 10.1021/jp205564n. Epub 2011 Nov 1.
Förster resonance energy-transfer (FRET) is a powerful and widely applied bioanalytical tool. According to the definition of FRET by Förster, for energy-transfer to take place, a substantial spectral overlap between the donor emission and acceptor excitation spectra is required. Recently also a phenomenon termed nonoverlapping FRET (nFRET) has been reported. The nFRET phenomenon is based on energy-transfer between a lanthanide chelate donor and a spectrally nonoverlapping acceptor and thus obviously differs from the conventional FRET, but the mechanism of nFRET and resulting implications to assay design have not been thoroughly examined. In this work, a homogeneous DNA-hybridization assay was constructed to study the distance and temperature dependency of both nFRET and conventional FRET. Capture oligonucleotides were labeled at the 5'-end with a Eu(III)-chelate, and these conjugates hybridized to complementary tracer oligonucleotides labeled with an organic fluorophore at various distances from the 3'-end. The distance dependency was studied with a fluorometer utilizing time-resolution, and the temperature dependency was studied using a frequency-domain (FD) luminometer. Results demonstrated a difference in both the distance and temperature dependency between conventional FRET and nFRET. On the basis of our measurements, we propose that in nFRET thermal excitation occurs from the lowest radiative state of the ion to a higher excited state that is either ionic or associated with a ligand-to-metal charge-transfer state.
Förster 共振能量转移（FRET）是一种强大且广泛应用的生物分析工具。根据 Förster 对 FRET 的定义，能量转移要发生，供体发射光谱和受体激发光谱之间需要有实质性的光谱重叠。最近也报道了一种称为非重叠 FRET（nFRET）的现象。nFRET 现象基于镧系螯合物供体和光谱非重叠受体之间的能量转移，因此明显不同于传统的 FRET，但 nFRET 的机制及其对分析设计的影响尚未得到彻底研究。在这项工作中，构建了一种均相 DNA 杂交测定法来研究 nFRET 和传统 FRET 的距离和温度依赖性。捕获寡核苷酸在 5'末端用 Eu(III)-螯合物标记，这些缀合物与从 3'末端以各种距离标记有有机荧光团的互补示踪寡核苷酸杂交。利用时间分辨率荧光计研究了距离依赖性，利用频域（FD）光度计研究了温度依赖性。结果表明，传统 FRET 和 nFRET 在距离和温度依赖性方面存在差异。基于我们的测量结果，我们提出在 nFRET 中，热激发从离子的最低辐射态发生到更高的激发态，该激发态要么是离子的，要么与配体-金属电荷转移态有关。
