Multiplexed DNA Origami-Based Biomarker Detection Assay for Early Dx of Arthritis

关节炎（AR）是美国残疾的主要原因，有5000万美国人（22%）患有某种形式的疾病或慢性关节症状。CDC估计，到2030年，将有6700万成年人患有AR，经济负担将达到每年1280亿美元的成本。虽然大多数AR病例被归类为骨关节炎（OA），影响超过2700万美国人，但其他形式的疾病也很普遍，包括类风湿性关节炎，青少年关节炎和痛风。AR与胶原蛋白的分解和炎症相关因子如细胞因子的产生有关。AR患者中I型和II型胶原蛋白的降解导致尿液中存在可检测的肽生物标志物;这些包括CTX-II、CTX-I和C2C。尿液中这些生物标志物的水平与患者软骨损伤的程度有关。骨关节炎生物标志物网络已根据其对OA诊断、预后和治疗效果的适用性对这些生物标志物进行了分类。由于AR进展缓慢，并且软骨变性患者通常多年无症状，因此在早期诊断AR并监测其进展一直是一个重大挑战。我们建议开发一种非侵入性的，高灵敏度的诊断检测AR使用DNA折纸分子，可以检测这三个生物标志物。在第一阶段，我们将评估针对CTX-II、CTX-I和C2C肽进行DNA折纸选择的可行性，以鉴定我们将在特异性诊断AR的测定中开发为探针的DNA序列。目标1：设计和构建一个用于AR生物标志物筛选的DNA折纸文库。目的2：分离与AR生物标志物肽相互作用的DNA折纸分子。目的3：鉴定和表征结合AR生物标志物的DNA折纸分子。由于DNA折纸分子结构，这些分子与AR生物标志物之间的相互作用应表现出增加的结合亲和力，这显著有助于该诊断测定的灵敏度和独创性。该检测将为医学界提供一种可靠的非侵入性AR检测方法，相对于目前的检测方法，该方法具有更高的灵敏度和便利性。在成功完成第一阶段目标后，这项工作将继续进入第二阶段，开发一种无标记、高灵敏度的检测方法，使用硅纳米线来发出尿液中这些生物标志物存在的信号。AR的非侵入性多重诊断测定的开发有可能导致临床范式从主要是姑息性的疗法转变为专注于识别早期AR患者的疗法。该测定还将具有用于监测接受各种治疗的患者中的软骨破坏的预后和治疗功效值。此外，该测定将提供必要的监测和极好的工具，以激励受影响的个体做出改变生活方式的决定（例如减肥），以提高他们的生活质量。最后，如果该方法的可行性得到证明，该方法可以应用于AR中的其他候选生物标志物。

DESCRIPTION (provided by applicant): Arthritis (AR) is the leading cause of disability in the United States, with 50 million Americans (22%) suffering from some form of the disease or chronic joint symptoms. The CDC estimates that, by the year 2030, 67 million adults will have AR and the economic burden will reach an annual cost of $128 billion dollars. While most AR cases are classified as osteoarthritis (OA), affecting over 27 million Americans, other forms of the disease are also prevalent, including rheumatoid arthritis, juvenile arthritis and gout. AR is associated with the breakdown of collagen and the production of inflammation-related factors such as cytokines. The degradation of Type I and Type II collagens in AR patients results in the presence of detectable peptide biomarkers in urine; these include CTX-II, CTX-I and C2C. The levels of these biomarkers in urine track with the extent of cartilage damage in patients. The Osteoarthritis Biomarkers Network has classified these biomarkers regarding their applicability to OA diagnosis, prognosis and efficacy of treatment. Because AR can progress slowly, and often patients with cartilage degeneration are asymptomatic for years, diagnosing AR in its early stages and monitoring its progression has been a major challenge. We propose to develop a non-invasive, highly sensitive diagnostic assay for AR using DNA origami molecules that can detect these three biomarkers. In Phase I, we will evaluate the feasibility of performing DNA origami selections against the CTX-II, CTX-I and C2C peptides to identify DNA sequences that we will develop as probes in an assay to specifically diagnose AR. Three aims are proposed: AIM1: Design and construct a DNA origami library for AR biomarker selections. AIM 2: Isolate DNA origami molecules that interact with AR biomarker peptides. AIM 3: Identify and characterize DNA origami molecules that bind the AR biomarkers. Due to the DNA origami molecular structure, the interaction between these molecules and the AR biomarkers should exhibit increased binding affinities, which contributes significantly to the sensitivity and ingenuty of this diagnostic assay. This assay will provide the medical community with a reliable, non-invasive test for AR that offers increased sensitivity and convenience relative to current assays. Following successful completion of our Phase I aims, this work will continue into Phase II, with the development of a label-free, highly sensitive detection assay, using silicon nanowires to signal the presence of these biomarkers in urine. The development of a non-invasive multiplex diagnostic assay for AR has the potential to result in a clinical paradigm shift from therapies tha are largely palliative to one that focuses on identifying persons with early stage AR. The assay will also have a prognostic and treatment efficacy value for monitoring cartilage breakdown in patients receiving various treatments. Further, this assay will provide the necessary surveillance and an excellent vehicle to motivate afflicted individuals to make lifestyle-changing decisions (e.g. weight loss) to increase their quality of life. Lastly, if the feasibility of the method is demonstrated, the method could be applied to other candidate biomarkers in AR.