Multiplexed mRNA quanitification using self-circularizing RNA probes

描述（由申请人提供）：对多种格式的敏感和准确的特定mrna水平测量方法的需求越来越大，同时这些方法快速且具有成本效益。例如，诸如sirna之类的基因靶向制剂需要能够监测预期靶标的特异性敲除以及对其他靶标的意外影响。的新兴领域？“治疗学”是将治疗学和诊断学结合起来，根据患者的基因型量身定制治疗，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，但目前用于测量生物标本中特定RNA水平的方法仍然存在技术局限性和潜在的偏差。基于目标扩增的方法，如Q-RT-PCR，虽然敏感且相当准确，但通常需要对每种分析物进行单独的反应，并且需要费力地分离细胞RNA和从DNA中纯化。另一组基于信号放大的方法可以避免纯化和复制目标序列，因此不太容易受到这些步骤中可能发生的偏差的影响。这些方法大多使用三明治杂交，这种方法速度慢，精度不高，也不是多路混合的最佳选择，因为分离非特异性杂交的均匀洗涤条件不能确保对富含AT和gc的目标进行无偏检测。

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.