Chib Rahul, Raut Sangram, Sabnis Sarika, Singhal Preeti, Gryczynski Zygmunt, Gryczynski Ignacy
Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107, USA ; Department of Physics and Astronomy, Texas Christian University, Fort Worth, TX, 76129, USA.
Methods Appl Fluoresc. 2014;2(1). doi: 10.1088/2050-6120/2/1/015003. Epub 2014 Jan 23.
Ethidium Bromide (EB) is a commonly used dye in a deoxyribonucleic acid (DNA) study. Upon an intercalation, this dye significantly increases its brightness and fluorescence lifetime. In this report we have studied the time-resolved fluorescence properties of EB existing simultaneously in free and DNA-bound forms in the solution. Fluorescence intensity decays were fitted globally to a double exponential model with lifetimes corresponding to free (1.6ns) and bound (22ns) forms, and molar fractions were determined for all used solutions. Anisotropy decays displayed characteristic time dependence with an initial rapid decline followed by recovery and slow decay. The short-lived fraction associated with free EB molecules decreases faster than long-lived fraction associated with EB bound to DNA. Consequently, contribution from fast rotation leads to initial rapid decay in anisotropy. On the other hand bound fraction, due to slow rotation helps recover anisotropy in time. This effect of associated anisotropy decays in systems such as EB free/EB-DNA is clearly visible in a wide range of concentrations, and should be taken into account in polarization assays and biomolecule dynamics studies.
溴化乙锭（EB）是脱氧核糖核酸（DNA）研究中常用的一种染料。插入后，这种染料会显著增加其亮度和荧光寿命。在本报告中，我们研究了溶液中以游离形式和与DNA结合形式同时存在的EB的时间分辨荧光特性。荧光强度衰减通过全局拟合为双指数模型，其寿命分别对应游离形式（1.6纳秒）和结合形式（22纳秒），并确定了所有使用溶液的摩尔分数。各向异性衰减呈现出特征性的时间依赖性，最初快速下降，随后恢复并缓慢衰减。与游离EB分子相关的短寿命部分比与结合到DNA上的EB相关的长寿命部分下降得更快。因此，快速旋转的贡献导致各向异性最初快速衰减。另一方面，由于旋转缓慢，结合部分有助于及时恢复各向异性。在广泛的浓度范围内，EB游离/EB-DNA等系统中相关各向异性衰减的这种效应清晰可见，在偏振分析和生物分子动力学研究中应予以考虑。
