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，全球人口的三分之一（总计约20亿）感染了结核分枝杆菌（TB）。大多数被感染的人（90％）永远不会收缩活跃的结核病，并且将保持无症状。但是，在免疫功能低下的患者中，结核病是全球死亡的主要原因。例如，结核病是HIV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（仅东南亚和非洲的600万人）与活跃结核病签约，并且超过100万人死于该疾病。预计今年有1000万次获得这种疾病，这是历史上最多的。许多结核病诊断测定正在市场上或开发。但是，大多数人不太适合在资源有限的设置中运行。他们总体上太昂贵，太耗时（24小时或更多），需要专业的设备，专业知识和功率，具有低敏感性（在痰涂片测试中为50％），或者不适用于HIV阳性患者或儿童（抗原测试）。据估计，更快，更准确的结核病诊断方法每年可以节省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环境中部署的新的传染病诊断测定法。我们寻求开发可以在实验室环境之外进行的测定方法，即没有PCR扩增，并且具有最低的力量，仪器和科学专业知识。我们建议通过将两种在Cu-Boulder修饰的DNA适体和“材料Dnazymes”结合使用的技术来实现这一目标 - 与基于电导的芯片平台相结合。事实证明，修饰的DNA适体对蛋白质靶标具有相似性和比抗体更好或更好的蛋白质靶标具有亲和力和特异性。但是，与抗体相比，DNA适体所需的时间和成本更少，并且更稳定。材料dnazymes是DNA序列，它们将原本稳定的金属复合体前体转化为金属纳米颗粒。该应用描述了使用改良的DNA适体/材料DNAZYME偶联物（材料aptazymes）用于尿液中TB脂肪团脂肪氨基氨基甘氨酸（LAM）的电检测。该项目的具体目标如下。目标1（1-6个月）。分离的TB LAM修饰的DNA适体。目标2（1-6个月）。分离的材料dnazymes介导含有[Au（cl）4] 1-的溶液中Au纳米颗粒的形成。目标3（6-12个月）。合成含有TB DNA适体和Au dnazyme的嵌合体，并验证每个序列是否正常。目标4（12-24个月）。确定模拟尿液中TB LAM的检测极限和特异性。这些目标完成后，我们将准备在一个RO1项目中使用该项目中开发的试剂和技术，该项目将验证尿液中的LAM作为结核病感染的生物标志物。