Osad'ko I S
Institute for Spectroscopy, RAS, 142190 Moscow, Russia.
J Chem Phys. 2015 Mar 28;142(12):125102. doi: 10.1063/1.4915279.
Possibility to create single donor-acceptor (D-A) pairs by attaching dye molecules to various sites of DNA strands with control of the inter-dye distance R enables one to measure average Förster resonance energy transfer (FRET) efficiency E as a function of R. Triplet states of the dyes influence the dependence E(R) considerably. Two types of FRET efficiency are considered: E = EA and E = ED. The efficiency EA(R) = JA(R)/[JA(R) + JD(R)] depends on the donor and the acceptor average intensities JD(R) and JA(R) measured in D- and A-fluorescence, whereas the efficiency ED(R) = 1 - JD(R)/JD(∞) depends only on the intensity of D-fluorescence, so-called the donor quenching method. The shape of the functions ED (R) and EA (R) depends strongly on whether the dyes have blinking fluorescence. FRET efficiencies ED (R) and EA (R) undergo the influence of many experimental factors and therefore, differ considerably from pure FRET efficiencies ED (s) (R) and EA (s) (R). Pure FRET efficiencies ED,A (s) (R) are calculated with the help of rate equations for D-A pairs, whose molecules have triplet states. It is shown how the calculated efficiencies ED,A (s) (R) can be compared to FRET efficiencies measured with the help of the intensities ID,A(R) corrected by cross talk and background light.
通过将染料分子连接到DNA链的不同位点并控制染料间距离R来创建单供体-受体（D-A）对的可能性，使得人们能够测量平均Förster共振能量转移（FRET）效率E作为R的函数。染料的三重态对E（R）的依赖性有很大影响。考虑了两种类型的FRET效率：E = EA和E = ED。效率EA（R）= JA（R）/[JA（R）+ JD（R）]取决于在供体和受体荧光中测量的供体和受体平均强度JD（R）和JA（R），而效率ED（R）= 1 - JD（R）/JD（∞）仅取决于供体荧光的强度，即所谓的供体猝灭法。函数ED（R）和EA（R）的形状强烈取决于染料是否具有闪烁荧光。FRET效率ED（R）和EA（R）受到许多实验因素的影响，因此与纯FRET效率ED（s）（R）和EA（s）（R）有很大差异。纯FRET效率ED，A（s）（R）是借助D-A对的速率方程计算得出的，其分子具有三重态。展示了如何将计算出的效率ED，A（s）（R）与借助串扰和背景光校正后的强度ID，A（R）测量的FRET效率进行比较。
