Department of Chemistry, University of Copenhagen, Copenhagen DK-2100, Denmark.
Nucleic Acids Res. 2013 Jan 7;41(1):e18. doi: 10.1093/nar/gks856. Epub 2012 Sep 12.
Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base-base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems.
Förster 共振能量转移（FRET）是一种常用于揭示生物分子结构和构象变化的技术，这些变化对所有生物都至关重要。通常，通过连接子将染料外部连接到核酸上，以实现 FRET，这使得实验的定量解释变得复杂，因为染料的扩散和重定向。在这里，我们报告了一种用于模拟和分析核酸中 FRET 的通用方法，该方法特别适用于对具有有限扩散和旋转自由度的探针之间的 FRET 进行建模，例如我们最近开发的碱基类似物 FRET 对（碱基对 FRET）。这些探针作为天然碱基之一的替代品位于 DNA/RNA 结构内部，从而可以独特地控制其位置和方向，并从感兴趣的内部位置进行报告。在不需要分子动力学建模的演示研究中，我们获得了以前无法获得的关于双链 DNA 中碱基的取向和纳秒动力学的见解，并重建了扭结 DNA 的高分辨率 3D 结构。所报告的方法伴随着一个免费的软件包 FRETmatrix，用于设计和分析含核酸系统中的 FRET。
