Fluorescence resonance energy transfer as a rich source of orientational information in nucleic acid structure

荧光光谱是生物系统中最灵敏的光谱分析方法，被广泛用于研究生物大分子的结构和动力学。荧光共振能量转移（FRET）被广泛用于测量或比较这些大分子，特别是核酸（DNA和RNA）中的距离。荧光是高度敏感的，因此它可以在单个分子的水平上进行研究，并且可以在细胞内进行。FRET是由连接在目标分子中已知位置的两个荧光基团之间的偶联产生的。这是通过空间，不需要物理接触。这个过程的效率取决于基团之间的距离，并且可以被布置在对于生物分子的研究最佳的长度范围内。在单分子的研究中，这可以用来分析分子的选定片段之间的距离，以及它如何在动态过程中作为时间的函数变化。这些信息在DNA和RNA结构的研究中特别有用。然而，一个众所周知的复杂性是FRET的大小取决于荧光基团的距离和方向。如果不正确对待后者，所获得的距离信息可能会非常误导。这严重限制了过去从FRET获得可靠的定量数据。另一方面，如果理解荧光基团的取向，这可以生成可靠的距离估计以及有价值的角度信息。该实验室最近的工作表明，当某些荧光基团（称为花青）用于核酸的FRET测量时，它们会堆叠在双螺旋的末端，并以确定的方式定向。我们已经使用这个属性来表明，我们可以从FRET数据测量角度信息，从而更可靠的距离信息。这使得FRET测量建立在可靠的定量基础上，我们现在希望将其开发为研究溶液中DNA和RNA结构的方法。这将对核酸的结构生物学和动力学性质产生重大影响。

Fluorescence is the most sensitive spectroscopic method used in biological systems, and is widely used to study the structure and dynamics of large biological molecules. Fluorescence resonance energy transfer (FRET) is extensively employed to measure or compare distances in such macromolecules, especially nucleic acids (DNA and RNA). Fluorescence is highly sensitive so that it can be studied at the level of a single molecule and it can be performed inside cells. FRET results from a coupling between two fluorescent groups attached at known positions in the molecule of interest. This is through space, not requiring physical contact. The efficiency of this process depends upon the distance between the groups, and can be arranged to be in the length range that is optimal for studies of biological molecules. In studies of single molecules this can be used to analyze distances between selected segments of a molecule, and how this changes as a function of time in dynamic processes. Such information is particularly powerful in the study of DNA and RNA structure. However, a well-known complication is that the magnitude of FRET depends on both distance and orientation of the fluorescent groups. If the latter is not properly treated the distance information obtained can be very misleading. This has severely limited obtaining reliable quantitative data from FRET in the past. On the other hand, if the orientation of the fluorescent groups is understood, this can generate reliable distance estimates together with valuable angular information. Recent work in this laboratory has demonstrated that when certain fluorescent groups (called cyanines) are used in FRET measurements in nucleic acids, they stack onto the ends of double helices where they are oriented in a defined manner. We have used this property to show that we can measure angular information from FRET data, and consequently more reliable distance information. This puts FRET measurements on a reliable quantitative basis and we now wish to develop this as a method to study the structures of DNA and RNA in solution. This should have a major impact in the structural biology and dynamic properties of nucleic acids.