Sensitive Detection of Viral Persistency Using Bioluminescent Stem-Loop Probes

 描述(由申请人提供)：RNA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、可直接监测各种基质的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，将是检测病毒持久性的理想方法，在这种情况下，病毒隐藏在特定的细胞类型中，但数量仍然很少。茎环探针是一类核酸生物传感器，是含有与靶序列互补的“环”区(约15-30个核苷酸)的单链寡核苷酸探针。这一“环”区域两侧有两个短的自互补区域(约5-7个核苷酸)，称为茎，通常与荧光团和猝灭剂结合。当互补靶点与环区杂交时，SLP从闭合构象转换为开放构象。在封闭构象中，荧光被能量传递机制猝灭，而在开放构象中，这种猝灭效应被消除，并且可以测量到荧光。这些探针的固有信号转导机制产生了优越的特异性和实时检测能力，而无需分离未杂交的探针。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能够作为低水平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同行的坚固的多功能性和广泛的适用性而提高当前基于SLP的检测系统的灵敏度，同时表现出低本底和高检测灵敏度。我们的假设将通过追求三个具体目标来验证，1)提高BSLP的分析性能和开发简便的BSLP合成方法，2)将具有高底物周转率以及耐热、长寿命、高活性的光蛋白的生物发光酶整合到BSLP设计中，3)设计、开发和优化BSLP，以艾滋病毒在细胞和血液样本中的持久性为模型系统来检测病毒的持久性。这项拟议的工作具有创新性，因为它引入了一种新的、新的传感策略，无缝地结合了生物发光蛋白的高灵敏度和SLP的特异性。这提供了一种快速检测任何样本基质中低水平病毒RNA的方法，例如在病毒持久性的情况下，解决了目前技术无法解决的问题。这项研究意义重大，因为它有望为核酸检测提供一种高度敏感的工具，从而能够检测病毒的持久性。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.