Multiplexed mRNA quanitification using self-circularizing RNA probes

描述（由申请人提供）：对用于以多重形式测量特定mRNA水平的灵敏和准确的方法的需求日益增加，该方法同时快速且具有成本效益。例如，基因靶向剂如siRNA需要监测预期靶标的特异性敲低以及对其他靶标的非预期效应的能力。新兴领域？”治疗诊断学“是指将治疗学和诊断学结合起来，根据患者的基因型进行治疗，并仔细监测治疗进展，特别需要能够准确地跟踪药物治疗对基因表达的影响，包括区分相关基因。尽管过去几年取得了进展，但目前用于测量生物样本中特定RNA水平的方法仍然存在技术限制和潜在偏差。基于靶扩增的方法，如Q-RT-PCR，虽然灵敏且相当准确，但通常需要对每种分析物进行单独的反应，以及费力地分离细胞RNA和从DNA纯化。另一组基于信号放大的方法可以避免靶序列的纯化和复制，因此不易于在这些步骤中发生偏差。这些方法中的大多数使用夹心杂交，这是缓慢的，不是非常准确的，并且对于多路复用不是最佳的，因为解离非特异性杂交体的均匀洗涤条件不能确保AT和GC富集靶的无偏检测。 我们提出了一种快速准确的mRNA定量的新方法，具有区分单核苷酸多态性（SNP）和易于复用的能力。该方法结合了成熟技术的元素，包括固相杂交和基于珠的多路复用，使用新的杂交探针，将发夹核酶。这些探针的靶向特异性文库，称为RNA Lassos，用于序列特异性结合细胞RNA样品中感兴趣的靶RNA并在其周围自环化，形成拓扑连接的复合物，该复合物抵抗我们用于消除背景杂交的严格洗涤条件。然后，通过使用标准的多路复用程序，对Lasso进行“解码”以量化与各种感兴趣的靶标互补的序列的存在。该方法不需要靶扩增，但允许信号扩增。该技术可用于任何感兴趣的基因组的高通量分析。作为第一个应用，我们计划开发用于临床样本中丙型肝炎病毒的同时定量和基因分型的检测方法，以帮助确定适当的治疗方案并跟踪治疗进展。该系统将特别适用于目前正在开发的用于包括HCV在内的几种疾病的基于RNAi的药物，其中监测靶标敲低和脱靶效应的能力很重要。

DESCRIPTION (provided by applicant): There is increasing demand for sensitive and accurate methods for measuring levels of specific mRNAs in multiplex format, which are at the same time fast and cost-effective. For example, gene-targeting agents such as siRNAs require the ability to monitor specific knock-down of the intended target as well as unintended effects on other targets. The emerging field of ?theranostics," the integration of therapeutics and diagnostics to tailor treatment to patient genotype and carefully monitor treatment progress, especially demands the ability to accurately follow the effects of drug treatment on gene expression including distinguishing related genes. Despite the progress of the last few years, current methods for measuring specific RNA levels in biological specimens still have technical limitations and potential biases. Methods based on target amplification, such as Q-RT-PCR, although sensitive and reasonably accurate, generally require a separate reaction for each analyte as well as laborious isolation of cellular RNA and purification from DNA. Another group of methods, based on signal amplification, can avoid the purification and replication of target sequences and hence is less prone to the biases that can occur during those steps. Most of these methods use sandwich hybridization, which is slow, not very accurate, and not optimal for multiplexing because uniform washing conditions to dissociate non-specific hybrids cannot ensure unbiased detection of both AT- and GC-rich targets. We propose a novel method for fast and accurate mRNA quantification with the ability to distinguish single-nucleotide polymorphisms (SNPs) and easy multiplexing. This method incorporates elements of proven technologies, including solid phase hybridization and bead-based multiplexing, with the use of novel hybridization probes that incorporate the hairpin ribozyme. Target-specific libraries of these probes, called RNA Lassos, are used to sequence-specifically bind to and self-circularize around target RNAs of interest within samples of cellular RNA, forming topologically-linked complexes that resist the stringent washing conditions we use to eliminate background hybridization. Lassos are then "decoded" to quantify the presence of sequences complementary to the various targets of interest by using standard multiplexing procedures. The method does not require target amplification, but permits signal amplification. This technology could be used for the high-throughput analysis of any set of genes of interest. As a first application, we plan to develop the assay for the simultaneous quantification and genotyping of hepatitis C virus in clinical samples, to help determine the appropriate treatment regimen and to follow treatment progress. This system will be particularly useful for RNAi-based drugs that are currently in development for several disorders, including HCV, where the ability to monitor target knock-down and off-target effects is important.