CAREER: Design and Characterization of Aptamer-Amphiphiles for Selective Binding

知识优势：适体是短单链寡核苷酸。它们是化学生产的，具有高亲和力和特异性结合各种不同靶点的能力，并且已被证明是有用的治疗剂和诊断工具。它们的亲和力通常与观察到的单克隆抗体相当，并且与肽相比显着更高。作为适配体-两亲体，适配体附着在疏水尾部，可以与其他两亲分子组装成各种不同的结构。它们与脂质组装形成适配体功能化脂质体将在生物技术应用中是理想的，例如靶向药物递送。PI建议将适配体作为两亲体进行研究，特别是结合fractalkine（仅在感染或炎症部位（如癌症）表达的粘附分子）的适配体序列，并假设适配体头组的方向以及疏水尾部和头组之间的间隔物的使用将影响分子的组装行为和适配体的二级结构。从而影响其对靶标的结合亲和力。为了验证这一假设，我们设置了以下研究任务：在研究任务1中，目标是测试设计的适配体-两亲体的生物学亲和力，在溶液中游离，并结合在脂质体中，从而评估哪些设计与fractalkine结合。在研究任务2中，PI将使用不同的技术（CD，熔化曲线，低温透射电镜，SAXS和SANS）来表征适配体-两亲体，以评估适配体的二级结构和两亲体的组装行为。这些结果将使适体两亲体的生物学功能与其结构和相行为之间的联系成为可能。这项研究将促进知识和理解，因为它不仅将为适配体-两亲体的设计提供有利的两亲性（头基取向、间隔设计、二级结构、组装行为），而且将提供对为什么这些性质是有益的基本理解。更广泛的影响：-技术影响。PI提出了一种新型工具的设计和表征，“适配体-两亲体”，基于通过改变其结构片段可以控制其形态，表面化学和功能的期望。本文首次提出了一种二级结构、两亲性和自组装性质的综合表征，以及这些性质与目标运载工具工程之间的联系。这些研究活动代表了变革性的研究，因为它们可能导致“适体-两亲体”（文献中的一个新名词）研究新领域的起源，该领域将融合合理的两亲体设计原则、化学、生物学和工程学，以提供靶向药物递送的先进系统。-社会/教育影响。Fractalkine是开发抗血管生成和抗癌药物的一个有吸引力的新靶点。通过选择与fractalkine结合的适配体作为模型系统，本研究概述了一种逻辑方法，可以应用于未来设计适配体-两亲体，这些适配体-两亲体可以功能化纳米粒子，提高粒子对fractalkine的特异性和亲和力，从而具有巨大的治疗潜力并显着造福社会。教学计划与研究计划紧密结合。PI正在与“DragonflyTV”（由美国国家科学基金会支持，由双城公共电视台（TPT）制作，并在全国公共广播服务（PBS）电视台播出的展示中学生做真实科学的科学系列节目）合作，目的是增进儿童和成人之间的科学和技术理解。此外，PI已经并将继续参加由TPT组织的旨在吸引女性从事科学和工程（PI的长期目标）的推广活动，目前正在与MRSEC合作，并将在未来继续这样做。通过MRSEC， PI将在夏季接待来自全国申请者的高中教师和少数族裔本科生，通过一个完善的高中网络，以及来自一些少数族裔服务机构的少数族裔本科生。

0846274KokkoliIntellectual Merit: Aptamers are short single-stranded oligonucleotides. They are chemically produced, have the capability of binding with high affinity and specificity a variety of different targets, and have been shown to be useful as therapeutic agents and diagnostic tools. Their affinities are often comparable to those observed for monoclonal antibodies, and are significantly higher compared to those of peptides. As aptamer-amphiphiles, aptamers attached to a hydrophobic tail, they can assemble with other amphiphilic molecules into a variety of different structures. Their assembly with lipids to form aptamer-functionalized liposomes will be desirable in biotechnological applications, such as, targeted drug delivery. The PI proposes to study aptamers as aptamer-amphiphiles and, in particular, aptamer sequences that bind fractalkine (an adhesion molecule expressed only at sites of infection or inflammation, such as cancer), and hypothesizes that the orientation of the aptamer headgroup, and the use of a spacer between the hydrophobic tail and the headgroup, will affect the assembly behavior of the molecule, and the secondary structure of the aptamer, thus subsequently affecting its binding affinity for its target. To test this hypothesis the following research tasks have been set: In Research Task 1, the goal is to test the biological affinity of the designed aptamer-amphiphiles, free in solution, and incorporated in liposomes, thus evaluating which designs bind to fractalkine. The PI will characterize the aptamer-amphiphiles in Research Task 2 with different techniques (CD, melting curves, cryo-TEM, SAXS, and SANS) that will evaluate the secondary structure of the aptamer, and assembly behavior of the amphiphiles. These results will enable a connection between the biological function of aptamer-amphiphiles with their structure and corresponding phase behavior. This research will advance knowledge and understanding as it will not only furnish favorable amphiphilic properties (headgroup orientation, spacer design, secondary structure, assembly behavior) for the design of aptamer-amphiphiles, but will provide a fundamental understanding of why these properties are beneficial. Broader Impact: -Technical Impact. The PI proposes the design and characterization of a novel tool, "aptamer-amphiphiles", based on the expectation that by changing their structural segments one can control their morphology, surface chemistry, and function. A comprehensive characterization of secondary structure, amphiphilic and self-assembly properties, and the link between these properties and the engineering of targeted delivery vehicles is proposed here for the first time. The research activities represent transformative research as they may lead to the genesis of a new field of research on "aptamer-amphiphiles" (a new term in the literature) that will merge rational amphiphile design principles, chemistry, biology, and engineering in order to provide advanced systems for targeted drug delivery. -Societal / Educational Impact. Fractalkine is an attractive new target for the development of anti-angiogenesis and anti-cancer agents. By choosing aptamers that bind to fractalkine as the model system, the proposed work outlines a logical approach that can be applied to the future design of aptamer-amphiphiles that can functionalize nanoparticles and increase the particle's specificity and affinity for fractalkine, thus having great therapeutic potential and significantly benefiting society. The educational plan is strongly integrated with the research plan. The PI is collaborating with "DragonflyTV" (a science series that presents middle school students doing real science, supported by NSF and produced by Twin Cities Public Television (TPT), and seen on Public Broadcasting Service (PBS) stations nationwide) with the goal of enhancing scientific and technological understanding among children and adults. Furthermore, the PI has participated, and continue to participate, in outreach activities organized by TPT with the goal of attracting women in sciences and engineering (a long term goal for the PI), and is currently collaborating, and will continue to do so in the future, with MRSEC. Through MRSEC the PI will be hosting during the summer high school teachers, and minority undergraduates from a nationwide pool of applicants, through a well established network of high schools, and minority undergraduates from a number of Minority Serving Institutions.