Multiplexed mRNA quanitification using self-circularizing RNA probes

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.