Modified DNA Aptamers and DNAzymes for Diagnosing TB in Resource-Poor Settings

描述(申请人提供)：据估计，全球人口的三分之一(总数约20亿)感染了结核分枝杆菌(TB)。大多数感染者(90%)将永远不会感染活动性结核病，并将保持无症状。然而，在免疫功能低下的患者中，结核病是全球范围内的主要死亡原因。例如，结核病是艾滋病毒阳性患者的主要死亡原因。世界卫生组织估计，2006年有900万人感染了活动性结核病(仅东南亚和非洲就有约600万人)，有100多万人死于这种疾病。预计今年还会有1000万人感染这种疾病，这是历史上感染人数最多的一年。许多结核病诊断化验方法已经上市或正在开发中。然而，大多数不太适合在资源有限的情况下运作。总的来说，它们太昂贵，太耗时(24小时或更长时间)，需要专门的设备、专业知识和电力，敏感性低(50%在痰涂片测试中)，或者对艾滋病毒阳性患者或儿童无效(抗原测试)。据估计，更快速、更准确的结核病诊断方法每年将挽救40万人的生命。这项研究的长期目标是开发可在资源有限的情况下部署的新的传染病诊断分析方法。我们寻求开发可以在实验室外进行的分析，即不需要PCR扩增，并且使用最小的功率、仪器和科学专业知识。我们建议通过将博尔德大学开发的两项技术--博尔德修饰的DNA适配子和“材料DNAzyme”--与基于电导的芯片平台相结合来实现这一目标。经过修饰的DNA适配子已被证明具有与抗体一样好或更好的蛋白质靶标的亲和力和特异性。然而，与抗体相比，DNA适配子需要更少的时间和成本来发现和生产，并且更具热稳定性。材料DNAzyme是将原本稳定的金属络合物前体转化为金属纳米颗粒的DNA序列。本申请描述了使用修饰的DNA适配子/材料DNAzyme结合物(材料aptazyme)来电检测尿液中的结核杆菌阿拉伯甘露聚糖(LAM)。该项目的具体目标如下。目标1(1-6个月)。分离结核分枝杆菌LAM修饰DNA适配子。目标2(1-6个月)。从含有[Au(Cl)4]1-的溶液中分离介导金纳米颗粒形成的材料DNAzyme。目标3(6-12个月)。合成含有TB DNA适配子和Au DNAzyme的嵌合体，并验证每个序列功能正常。目标4(12-24个月)。确定模拟尿液中结核LAM的检出限和特异性。在完成这些目标后，我们将准备将在这个项目中开发的试剂和技术用于RO1项目，该项目将验证尿液中的LAM作为结核病感染的生物标志物。

DESCRIPTION (provided by applicant): It has been estimated that one-third of the global population (total of ~2 billion) is infected with Mycobacterium tuberculosis (TB). A majority of those infected (90%) will never contract active TB and will remain asymptomatic. In immunocompromised patients, however, TB is a major cause of death worldwide. For example, TB is the leading cause of death in HIV-positive patients. The World Health Organization estimates that 9 million people contracted active TB in 2006 (~6 million from Southeast Asia and Africa alone) and over 1 million died from the disease. Another 10 million are projected to acquire the disease this year, the most in history. Many TB diagnostic assays are on the market or in development. A majority, however, are not well suited for operation in resource-limited settings. Collectively they are too expensive, too time consuming (24 hrs or more), require specialized equipment, expertise, and power, have low sensitivities (50% in the sputum smear test), or do not work on HIV-positive patients or children (antigen tests). It has been estimated that more rapid and accurate TB diagnostic methods would save 400,000 lives per year. The long-term goal of this research is to develop new infectious disease diagnostic assays that can be deployed in resource-limited settings. We seek to develop assays that can be performed outside of a laboratory setting, that is, without PCR amplification, and with minimal power, instrumentation, and scientific expertise. We propose to accomplish this goal by combining two technologies developed at CU-Boulder-modified DNA aptamers and "materials DNAzymes"-with a conductance-based chip platform. Modified DNA aptamers have proven to have affinities and specificities for protein targets that are as good as or better than antibodies. Compared to antibodies, however, DNA aptamers require less time and cost to discover and produce, and are more thermally stable. Materials DNAzymes are DNA sequences that convert otherwise stable metal complex precursors into metal nanoparticles. This application describes the use of modified DNA aptamer/materials DNAzyme conjugates (materials aptazymes) for the electrical detection of TB lipoarabinomannan (LAM) from urine. The Specific Aims for the project are as follows. Aim 1 (Months 1-6). Isolate modified DNA aptamers for TB LAM. Aim 2 (1-6 months). Isolate materials DNAzymes that mediate the formation of Au nanoparticles from solutions containing [Au(Cl)4]1-. Aim 3 (Months 6-12). Synthesize a chimera containing a TB DNA aptamer and Au DNAzyme and verify that each sequence functions properly. Aim 4 (Months 12-24). Determine detection limits and specificities of TB LAM in simulated urine. Upon completion of these aims, we will be poised to use the reagents and technology developed in this project in an RO1 project that will validate LAM in urine as a biomarker of TB infection.