Sensitive Detection of Viral Persistency Using Bioluminescent Stem-Loop Probes

 描述（由申请方提供）：RNA的检测可用于微生物的快速鉴定、基因调控分析、分子诊断和病毒持久性检测。然而，仍然需要快速、定量、灵敏并且适合于在各种基质中直接监测的RNA检测方法。本文介绍的工作描述了一种满足这些特征的方法，并且将是检测病毒持续存在的理想方法，在这种情况下，病毒隐藏在特定的细胞类型中，而数量很少。 茎环探针（SLP）是一类核酸生物传感器，其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"区域的侧翼是两个短的自身互补区域（约5 - 7nt），称为茎，其通常与荧光团和猝灭剂缀合。在互补靶与环区域杂交后，SLP从闭合构象转换为开放构象。在闭合构象中，荧光被能量转移机制猝灭，而在开放构象中，这种猝灭效应被去除，并且可以测量荧光。这些探针的固有信号转导机制产生上级特异性和在不分离未杂交探针的情况下进行真实的检测的能力。然而，目前可用的SLP具有有限的灵敏度。解决目前SLP中存在的这些问题并产生有效的解决方案将使SLP能够作为低水平RNA检测的平台，这在病毒持续存在的情况下是必要的。为此，我们假设，生物发光SLP（BSLP）的发展将提高目前的SLP为基础的检测系统的灵敏度，通过保留其荧光同行的坚固的通用性和广泛的适用性，同时表现出低背景和高检测灵敏度。 我们的假设将通过追求三个具体目标进行测试，1）提高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性，长寿命，高活性的光蛋白纳入BSLP设计中，3）设计，开发和优化BSLP，用于使用HIV持续存在作为细胞和血液样本中的模型系统检测病毒持续存在。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的特异性无缝结合在一起。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，因为它有望为检测核酸提供一种高度灵敏的工具，从而可以检测病毒的持续性。这反过来将对传染病诊断和治疗监测产生重大影响。

 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.