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

KokkoliIntelligence优点：适配子是短的单链寡核苷酸。它们是化学产生的，具有高亲和力和特异性结合各种不同靶点的能力，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单抗的亲和力相当，而与多肽相比明显更高。作为适配子-两亲分子，连接在疏水尾巴上的适配子可以与其他两亲性分子组装成各种不同的结构。它们与脂类组装形成适体功能化脂质体在生物技术应用中是可取的，例如靶向药物输送。PI建议将适体作为适体-双亲性分子来研究，特别是与Fractalkine(一种仅在感染或炎症部位表达的黏附分子，如癌症)结合的适体序列，并假设适体头基的取向以及疏水尾部和头基之间的间隔基的使用将影响分子的组装行为和适体的二级结构，从而影响其与靶标的结合亲和力。为了验证这一假设，已经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体-两亲分子的生物亲和力，这些两亲性分子在溶液中游离，并被结合到脂质体中，从而评估哪些设计与Fractalkine结合。PI将使用不同的技术(CD、熔化曲线、低温电子显微镜、SAXS和SANS)来表征研究任务2中的适体-两亲分子，以评估适体的二级结构和两亲分子的组装行为。这些结果将使适体-两亲分子的生物学功能与其结构和相应的相行为之间建立联系。这项研究将增进人们的认识和理解，因为它不仅将为适体-两亲分子的设计提供良好的两亲性(头基取向、间隔基设计、二级结构、组装行为)，而且将从根本上理解为什么这些性质是有益的。更广泛的影响：-技术影响。PI提出了一种新的工具--适体-两亲性的设计和表征，基于这样的期望，即通过改变它们的结构段可以控制它们的形态、表面化学和功能。首次提出了二级结构、两亲性和自组装性质的综合表征，以及这些性质与靶向递送载体工程之间的联系。这些研究活动代表着变革性的研究，因为它们可能导致一个新的研究领域的起源，即“适体-两亲性”(文献中的一个新术语)，它将融合合理的两亲性设计原则、化学、生物学和工程学，以便为靶向药物输送提供先进的系统。-社会/教育影响。Fractalkine是抗血管生成和抗癌药物开发的一个有吸引力的新靶点。通过选择与Fractalkine结合的适配子作为模型系统，所提出的工作概述了一种合理的方法，可以应用于未来的适配子-两亲分子的设计，这种适配子-两亲分子可以使纳米粒子功能化，增加颗粒对Fractalkine的特异性和亲和力，从而具有巨大的治疗潜力，并显著造福社会。教育计划与研究计划紧密结合。该协会正在与“龙飞电视”合作(这是一部由美国国家科学基金会支持、双城公共电视台(TPT)制作、在全国公共广播服务(PBS)电视台播出的科学连续剧，讲述中学生做真正的科学)，目的是增进儿童和成年人对科学和技术的理解。此外，私营部门已经并将继续参加技术合作项目组织的外联活动，目的是吸引妇女从事科学和工程工作(私营部门的一个长期目标)，目前正在并将继续与MRSEC合作。通过MRSEC，PI将在暑期接待高中教师和全国范围内申请者中的少数族裔本科生，通过建立良好的高中网络，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.