Sensitive Detection of Viral Persistency Using Bioluminescent Stem-Loop Probes

使用生物发光茎环探针灵敏检测病毒持久性

• 批准号: 9096837
• 负责人: Sapna K Deo
• 金额: $ 29.55万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9096837
• 关键词: Address; Area; Biochemical; Biological Assay; Biological Models; Biosensor; Blood specimen; Body Fluids; Cells; Characteristics; Chemicals; Chickenpox; Communicable Diseases; DNA; Data; Detection; Development; Diagnostic; Employment; Energy Transfer; Enzymes; Exhibits; Fluorescence; Formulation; Gene Expression Regulation; Generations; Genetic Variation; Goals; HIV; Health; Herpes zoster disease; In Vitro; Infection; Life; Light; Luminescent Proteins; Measles; Measurement; Measures; Methods; Modeling; Molecular Conformation; Monitor; Noise; Nucleic Acids; Nucleotides; Oligonucleotide Probes; Oligonucleotides; Performance; Protein Engineering; Proteins; Qualifying; RNA; Reporter; Research; Research Personnel; Sampling; Signal Transduction; Simplexvirus; Single Nucleotide Polymorphism; Source; Specificity; System; Technology; Testing; Therapeutic; Time; Viral; Virus; Work; analytical method; base; cell type; clinical application; design; experience; fluorophore; improved; in vivo; innovation; microorganism; molecular diagnostics; novel; nucleic acid detection; rapid detection; rapid technique; screening; small molecule; stem; tool; viral RNA; viral detection

 DESCRIPTION (provided by applicant): The detection of RNA provides for rapid identification of microorganisms, gene regulation analysis, molecular diagnostics, and detection of viral persistency. However, there is still a need for RNA detection methods that are rapid, quantitative, sensitive, and amenable for direct monitoring in a variety of matrices. The work presented within describes a method that fulfills these characteristics, and will be ideal for the detection of viral persistency, a scenario in which viruses hide within a specific cell type while remaining few in number. Stem-loop probes (SLPs) are a class of nucleic acid biosensors that are single-stranded oligonucleotide probes containing a "loop" region (about 15-30 nt) complementary to a target sequence. This "loop" region is flanked by two short self-complementary regions (about 5-7 nt) known as the stem, which are typically conjugated to a fluorophore and a quencher. The SLP switches from closed to open conformations upon hybridization of a complementary target to the loop region. In the closed conformation, fluorescence is quenched by an energy transfer mechanism, whereas in the open conformation this quenching effect is removed, and fluorescence can be measured. The inherent signal transduction mechanism of these probes yields superior specificity and the ability to perform detection in real time without separation of unhybridized probes. However, currently available SLPs have limited sensitivity. Addressing these problems present in current SLPs and generating effective solutions will allow SLPs to serve as a platform of low-level RNA detection, as is necessary in the case of viral persistency. Toward that end, we hypothesize that the development of a bioluminescent SLP (BSLP) would enhance sensitivity of current SLP-based detection systems by retaining the rugged versatility and broad applicability of its fluorescent counterparts while exhibiting low background and high detection sensitivity. Our hypothesis will be tested by pursuing three specific aims, 1) Enhance analytical performance of BSLPs and develop facile BSLP synthesis methods, 2) Incorporate bioluminescent enzymes possessing high substrate turnover as well as thermostable, long-lived, highly active photo proteins into BSLP design, 3) Design, develop and optimize BSLPs for the detection of viral persistency using HIV persistency as a model system in cell and blood samples. The proposed work is innovative because it introduces a novel, new sensing strategy that seamlessly combines the high sensitivity of bioluminescent proteins and the specificity of SLPs. This provides a means for the rapid detection of low levels of viral RNA in any sample matrix such as would exist in the case of viral persistency, solving a problem current technologies aren't capable of addressing. This research is significant because it is expected to provide a highly sensitive tool for the detection of nucleic acids that will allow for the detection of viral persistency. This, in turn, wll have a significant impact on infectious disease diagnostics and therapeutic monitoring.

 描述（由申请人提供）：RNA的检测提供了微生物的快速鉴定、基因调控分析、分子诊断和病毒持久性的检测。然而，仍然需要快速、定量、灵敏且适合在各种基质中直接监测的 RNA 检测方法。其中介绍的工作描述了一种满足这些特征的方法，并且非常适合检测病毒持久性，即病毒隐藏在特定细胞类型中而数量很少的情况。 茎环探针 (SLP) 是一类核酸生物传感器，是含有与靶序列互补的“环”区域（约 15-30 nt）的单链寡核苷酸探针。该“环”区域的两侧是两个短的自互补区域（约 5-7 nt），称为茎，通常与荧光团和猝灭剂缀合。当互补靶标与环区域杂交时，SLP 从闭合构象转变为开放构象。在闭合构象中，荧光通过能量转移机制被猝灭，而在开放构象中，这种猝灭效应被消除，并且可以测量荧光。这些探针固有的信号转导机制具有卓越的特异性，并且能够在不分离未杂交探针的情况下进行实时检测。然而，目前可用的 SLP 的灵敏度有限。解决当前 SLP 中存在的这些问题并产生有效的解决方案将使 SLP 能够充当低水平 RNA 检测的平台，这在病毒持久性的情况下是必要的。为此，我们假设生物发光 SLP (BSLP) 的开发将通过保留其荧光对应物的坚固多功能性和广泛适用性，同时表现出低背景和高检测灵敏度，来提高当前基于 SLP 的检测系统的灵敏度。 我们的假设将通过追求三个具体目标来检验，1）增强 BSLP 的分析性能并开发简便的 BSLP 合成方法，2）将具有高底物周转率的生物发光酶以及热稳定、长寿命、高活性的光蛋白纳入 BSLP 设计，3）设计、开发和优化 BSLP，以使用 HIV 持久性作为模型来检测病毒持久性 细胞和血液样本中的系统。这项工作具有创新性，因为它引入了一种新颖的传感策略，将生物发光蛋白的高灵敏度和 SLP 的特异性无缝结合起来。这提供了一种快速检测任何样品基质中低水平病毒RNA的方法，例如在病毒持续存在的情况下，解决了当前技术无法解决的问题。这项研究意义重大，因为它有望提供一种高度灵敏的核酸检测工具，从而可以检测病毒的持久性。反过来，这将对传染病诊断和治疗监测产生重大影响。