CAREER: Structure-Specific Fluorescence Spectroscopy to Dissect Conformational Heterogeneity in Macromolecules

在化学系化学测量和成像计划的支持下，以及分子和细胞生物科学系分子生物物理组的部分共同资助下，俄勒冈大学(UO)的Julia Widom博士和她的研究小组正在开发测量和分析方法，以高空间和时间分辨率解析生物大分子采用的各种结构。生物大分子经常跨越多个构象结构，这些结构的性质和普遍存在对于它们的内在生物学功能以及它们作为药物靶标、生物标志物和纳米结构构建块的潜在应用都具有重要意义。以RNA和DNA为重点，Widom实验室正在建立基于高分辨率激光光谱学的方法，从不同结构的大分子中提取不同的特征，并正在扩展可用于此类测量的具有良好特征的分子探测器工具箱。此外，Widom博士正在与UO本科生化学俱乐部合作，通过远程互动化学活动、农村学生对UO的现场访问以及与东俄勒冈大学的化学项目合作，参与到俄勒冈州农村社区的初中生和高中生中。大多数现有的光谱技术对被测量系统的局部(Angstrom长度尺度)或全球(纳米长度尺度)结构敏感。为了克服这一限制，Widom博士的研究小组正在开发一种基于超快时间尺度上的荧光光谱测量的方法，利用两个探针之间的激发态能量转移来从紧密和松散折叠的构象亚群中分离出信号。得到的时间分辨发射光谱的全局拟合将两个探测器的局部环境与它们所在的大分子的全局构象联系起来。具有至少两个明确全球构象的结构化DNA和RNA低聚物将被用作模型系统，随后将扩展技术以研究小分子与RNA的结合。Widom实验室还将调查不同荧光探针在涉及核酸的应用中的适用性，分析它们的光物理性质及其对RNA结构的影响。如果成功，这个项目有可能为分析复杂的化学、生物和材料系统的结构异质性提供新的工具，从而产生深远的科学和更广泛的影响。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, and partial co-funding from the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences, Dr. Julia Widom and her research group at the University of Oregon (UO) are developing measurement and analysis methods to resolve with high spatial and temporal resolution the diverse structures adopted by biological macromolecules. Biological macromolecules frequently transition across multiple conformational structures, and the nature and prevalence of these structures is of great significance to both their intrinsic biological functions, and their potential applications as drug targets, biomarkers, and building blocks in nanostructures. With a focus on RNA and DNA, the Widom Lab is establishing methods based on high-resolution laser spectroscopy that extract distinct signatures from macromolecules with different structures, and is expanding the toolbox of well-characterized molecular probes that can be used for such measurements. In addition, Dr. Widom is partnering with the UO undergraduate Chemistry Club to engage in outreach to middle- and high-school students in rural Oregon communities through remote, interactive chemistry activities, site visits to UO by rural students, and collaboration with the chemistry program at Eastern Oregon University.Most existing spectroscopic techniques are sensitive to either the local (Angstrom length-scale) or global (nanometer length-scale) structure of the system being measured. To overcome this limitation, Dr. Widom’s research group is developing a method based on measurement of fluorescence spectra on an ultrafast timescale, utilizing excited-state energy transfer between two probes to separate signals from tightly and loosely folded conformational subpopulations. Global fitting of the resulting time-resolved emission spectra connects the local environments of the two probes to the global conformations of the macromolecule within which they reside. Structured DNA and RNA oligomers with at least two well-defined global conformations will be used as model systems, followed by expansion of the technique to study the binding of small molecules to RNA. The Widom Lab will also investigate the suitability of different fluorescent probes for applications involving nucleic acids, analyzing their photophysical properties and their impacts on RNA structure. If successful, this project has the potential to provide new tools for the analysis of structural heterogeneity in complex chemical, biological and material systems and thus have far-reaching scientific broader impact.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.