Multiplexed mRNA quanitification using self-circularizing RNA probes

描述(由申请人提供)：对以多重格式测量特定mRNAs水平的灵敏和准确的方法的需求日益增加，这些方法同时具有快速和成本效益。例如，基因靶向剂，如siRNAs，需要能够监测预期目标的特定击倒以及对其他目标的意外影响。治疗学是一个新兴的领域，它将治疗学和诊断学相结合，根据患者的基因类型量身定做治疗方案，并仔细监测治疗进展，尤其要求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，但目前测量生物标本中特定RNA水平的方法仍然存在技术限制和潜在的偏差。基于靶扩增的方法，如Q-RT-PCR，虽然敏感和相当准确，但通常需要对每个分析物进行单独的反应，以及费力地分离细胞RNA和从DNA中纯化。另一组基于信号放大的方法可以避免目标序列的纯化和复制，因此不太容易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，这种方法速度慢，不是很准确，而且对于多重来说也不是最佳的，因为均匀的洗涤条件来分离非特异性杂交不能确保对富含AT和GC的靶标的无偏见检测。 我们提出了一种新的快速、准确的mRNA定量方法，具有区分单核苷酸多态(SNPs)的能力和易于多重的能力。这种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路复用，并使用了含有发夹状核酶的新型杂交探针。这些探针的目标特异性文库被称为RNA Lassos，用于与细胞RNA样本中感兴趣的目标RNA进行序列特异性结合和自我循环，形成拓扑连接的复合体，从而抵抗我们用来消除背景杂交的严格洗涤条件。然后，通过使用标准的多路传输程序，对套索进行“解码”，以量化与各种目标互补的序列的存在。该方法不需要目标放大，但允许信号放大。这项技术可以用于对任何一组感兴趣的基因进行高通量分析。作为第一个应用，我们计划开发一种同时对临床样本中的丙型肝炎病毒进行定量和基因分型的分析方法，以帮助确定适当的治疗方案并跟踪治疗进展。该系统将对基于RNAi的药物特别有用，这些药物目前正在开发中，用于治疗几种疾病，包括丙型肝炎病毒，在这些药物中，监测靶向击倒和脱靶效应的能力是重要的。

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.