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

用于在资源匮乏环境中诊断结核病的修饰 DNA 适体和脱氧核糖核酸酶

• 批准号: 8431996
• 负责人: DAN L. FELDHEIM
• 金额: $ 17.87万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8431996
• 关键词: Affinity; Africa; Antibodies; Antigens; Aptamer Technology; Binding; Biological Assay; Biological Markers; Catalytic DNA; Cause of Death; Chemistry; Child; Chimera organism; Clinical; Colorado; Communicable Diseases; Contracts; DNA; DNA Sequence; Detection; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Disease; Electrodes; Enzymes; Equipment; Goals; Gold; HIV Seropositivity; Immunocompromised Host; Infection; Ions; Laboratories; Marketing; Measures; Mediating; Metals; Methods; Mycobacterium tuberculosis; Patients; Plasma; Population; Proteins; RNA Sequences; Reagent; Recording of previous events; Refrigeration; Reporting; Research; Resources; Sampling; Serum; Simulate; Solutions; Southeastern Asia; Specificity; Sputum; Surface; Technology; Testing; Time; Tuberculosis; Universities; Urine; Work; World Health Organization; aptamer; base; cost; instrumentation; lipoarabinomannan; metal complex; nanoparticle; novel diagnostics; operation; professor

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.

描述（由申请人提供）：据估计，全球三分之一的人口（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，并且将保持无症状。然而，在免疫功能低下的患者中，结核病是世界范围内死亡的一个主要原因。例如，结核病是艾滋病毒阳性患者死亡的主要原因。世界卫生组织估计，2006年有900万人感染活动性结核病（仅东南亚和非洲就有约600万人），100多万人死于该病。预计今年还会有1000万人感染这种疾病，这是历史上最多的。许多结核病诊断检测方法已经上市或正在开发中。但是，大多数方法不太适合在资源有限的情况下进行操作。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（痰涂片试验50%），或对艾滋病毒阳性患者或儿童不起作用（抗原试验）。据估计，更快速和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以在资源有限的环境中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环境之外进行的检测，即无需PCR扩增，并且具有最小的功率，仪器和科学专业知识。为了实现这一目标，我们建议将科罗拉多大学博尔德分校开发的两种技术——修饰DNA适体和“材料DNAzymes”——与基于电导的芯片平台结合起来。经过修饰的DNA适体已被证明对蛋白质目标具有亲和力和特异性，与抗体一样好，甚至更好。然而，与抗体相比，DNA适体需要更少的时间和成本来发现和生产，并且更热稳定。DNAzymes是一种DNA序列，可以将其他稳定的金属复合物前体转化为金属纳米颗粒。本申请描述了使用修饰的DNA适体/材料DNAzyme偶联物（材料适体酶）电检测TB脂阿拉伯糖甘露聚糖（LAM）从尿液。该项目的具体目标如下。目标1（1-6个月）。分离TB LAM修饰DNA适体。目标2（1-6个月）。从含有[Au(Cl)4]1-的溶液中分离出介导金纳米颗粒形成的物质DNAzymes。目标3（6-12个月）。合成含有结核DNA适体和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，我们将准备在RO1项目中使用该项目开发的试剂和技术，该项目将验证尿液中的LAM作为结核病感染的生物标志物。