Multiplexed mRNA quanitification using self-circularizing RNA probes

描述（由申请人提供）：对于以多重形式测量特定 mRNA 水平的灵敏且准确的方法的需求不断增加，同时该方法快速且具有成本效益。例如，siRNA 等基因靶向剂需要能够监测预期靶标的特异性敲低以及对其他靶标的意外影响。新兴的“治疗诊断学”领域，即治疗学和诊断学的整合，以根据患者基因型定制治疗并仔细监测治疗进展，特别需要能够准确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，但目前测量生物样本中特定 RNA 水平的方法仍然存在技术局限性和潜在偏差。基于靶标扩增的方法，例如 Q-RT-PCR，虽然灵敏且相当准确，但通常也需要对每种分析物进行单独的反应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，因此在这些步骤中不太容易出现偏差，这些方法大多数使用夹心杂交，这种方法速度慢，不太准确，并且对于多重分析来说不是最佳的，因为解离非特异性杂交体的统一洗涤条件不能确保对富含 AT 和 GC 的靶标进行公正的检测。 我们提出了一种快速准确的 mRNA 定量新方法，能够区分单核苷酸多态性 (SNP) 且易于多重分析。该方法结合了成熟技术的元素，包括固相杂交和基于珠子的多重技术，并使用包含发夹核酶的新型杂交探针。这些探针的目标特异性文库称为 RNA Lassos，用于序列特异性地结合细胞 RNA 样品中感兴趣的目标 RNA 并在其周围自环化，形成拓扑连接的复合物，可抵抗我们用于消除背景杂交的严格洗涤条件。然后使用标准多重程序对套索进行“解码”，以量化与各种感兴趣的靶标互补的序列的存在。该方法不需要靶标放大，但允许信号放大。该技术可用于任何感兴趣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同步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。该系统对于目前正在开发的针对多种疾病（包括 HCV）的基于 RNAi 的药物特别有用，其中监测目标敲低和脱靶效应的能力非常重要。

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.