Investigating Conformations of Single Polysaccharides and Nucleic Acids by Force Spectroscopy

该项目的重点是利用原子力显微镜（AFM）技术研究多糖和单链DNA的强制构象转变。目的是：1)利用力钳AFM监测海藻酸盐的外显异构反应；2)改进和扩展糖弹性的计算机建模；3)利用力谱法研究单链核酸的弹性及其碱基叠加相互作用。为了实现这些目标，藻酸盐前体将在C-5外甲酰基酶存在的AFM中拉伸，甘露醛酸到古鲁醛酸的构象转化将通过力钳AFM监测。糖的弹性将采用计算方法建模，以解决两个重要问题：糖中原子相互作用能的准确性以及模拟和AFM实验之间的时间差距。原子力显微镜将测量单个同源多核苷酸的弹性，并在不同溶剂条件下建立碱基之间堆叠相互作用的特征。该项目将推进单分子生物物理学研究，并将增加对多糖和单链DNA的机械化学的理解。该项目还将开发新的方法来模拟过滤糖。测量碱基堆叠相互作用中涉及的力对理解DNA和RNA的结构和功能具有重要意义。该项目将为两名研究生和两名本科生提供令人兴奋的教育和研究机会，并将有利于碳水化合物建模的研究人员。外展活动将涉及本科生，K12学生和他们的老师。该项目由生物科学理事会分子与细胞生物科学部的分子生物物理学和数学与物理科学理事会化学部的实验物理化学项目共同支持。

The project focuses on investigating forced conformational transitions in polysaccharides and single-stranded DNA using atomic force microscopy (AFM) techniques. The objectives are: 1) Use force-clamp AFM to monitor the epimerization reaction in alginates; 2) Improve and expand computer modeling of sugars' elasticity; 3) Use force spectroscopy to examine the elasticity of single stranded nucleic acids and their base stacking interactions. To achieve these objectives alginate precursors will be stretched in an AFM in the presence of C-5 epimerase enzyme and the conformational conversion of the mannuronic acid to guluronic acid will be monitored by force-clamp AFM. Sugars' elasticity will be modeled with computational methods developed to address two important issues: the accuracy of the atomic interaction energy in sugars and the time gap between simulations and AFM experiments. The elasticity of individual homo-polynucleotides will be measured by AFM and the signatures of stacking interactions among the bases will be established under various solvent conditions. The project will advance single-molecule biophysics research and will increase the understanding of the mechanochemistry of polysaccharides and single-stranded DNA. The project will also develop novel approaches to modeling strained sugars. Measurements of forces involved in base stacking interactions are of a fundamental significance to the understanding of DNA and RNA structure and function. This project will provide an exciting education and research opportunity for two graduate and two undergraduate students and will benefit researchers modeling carbohydrates. Outreach activities will involve undergraduate students, K12 students and their teachers. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Experimental Physical Chemistry Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.