Marini Monica, Falqui Andrea, Moretti Manola, Limongi Tania, Allione Marco, Genovese Alessandro, Lopatin Sergei, Tirinato Luca, Das Gobind, Torre Bruno, Giugni Andrea, Gentile Francesco, Candeloro Patrizio, Di Fabrizio Enzo
SMILEs Lab, Physical Science and Engineering (PSE) and Biological and Environmental Science and Engineering (BESE) Divisions, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.
NABLA Lab, BESE Division, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.
Sci Adv. 2015 Aug 28;1(7):e1500734. doi: 10.1126/sciadv.1500734. eCollection 2015 Aug.
The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.
DNA的结构于1953年通过X射线纤维衍射得以确定。人们已多次尝试用其他技术获取DNA的直接图像。直接成像旨在对分子中所有相关特征长度进行定量评估。DNA的直接图像与倒易空间中的衍射不同，难以获得主要有两个原因：构成分子的元素固有的极低对比度，以及在保持样品原始形状和大小的同时制备样品的困难。我们表明，通过与DNA生理条件兼容的制备程序，可以获得单个悬浮DNA分子的直接图像。在图像中，A-DNA的所有相关长度均可测量。本实验使用了一台在80 keV下运行、极限分辨率为1.5 Å的高分辨率透射电子显微镜。鉴于通过晶体或纤维的X射线衍射获得的平均信息不足以进行详细的结构测定，或者当无法从生物分子中获得晶体或不足以理解多种蛋白质构型时，单个分子的直接成像可作为一种解决需要单分子水平知识的生物学问题的方法。
