Multiplexed mRNA quanitification using self-circularizing RNA probes

使用自环化 RNA 探针进行多重 mRNA 定量

• 批准号: 7463751
• 负责人: SERGEI A KAZAKOV
• 金额: $ 30万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7463751
• 关键词: Affect; Affinity; Aftercare; Alkalies; Binding; Biological; Biological Assay; Blood; Blood capillaries; Blood specimen; Cantor; Catalytic RNA; Cell Extracts; Cells; Chemicals; Chronic Hepatitis C; Clinical; Code; Collection; Color; Complex; Condition; DNA; DNA Polymerase Inhibitor; DNA Primers; DNA Probes; DNA purification; Data; Detection; Development; Diagnostic; Disease; Disease Progression; Dissociation; Dot Immunoblotting; Elements; Ensure; Figs - dietary; Gene Expression; Gene Targeting; Generations; Genes; Genotype; Guanine + Cytosine Composition; H-DNA; Hepatitis C; Hepatitis C virus; Hereditary Disease; Hybrids; In Vitro; Internal Ribosome Entry Site; Kinetics; Label; Lasso; Libraries; Link; Liver; Measures; Mediator of activation protein; Membrane; Messenger RNA; Methods; Modeling; Monitor; Noise; Nylons; Oligonucleotides; Patients; Peptide Signal Sequences; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Polymerase Chain Reaction; Primer Extension; Procedures; Production; Property; RNA Interference; Reaction; Research; Research Design; Reverse Transcription; Ribonuclease H; Sampling; Scheme; Signal Transduction; Single Nucleotide Polymorphism; Site; Small Interfering RNA; Solid; Solutions; Specificity; Specimen; Speed; Standards of Weights and Measures; Structure; Surface; System; TNF gene; Technology; Testing; Therapeutic; Time; Treatment Protocols; Tube; base; capillary; cost; crosslink; cytokine; design; design and construction; hairpin ribozyme; high throughput analysis; improved; interest; knock-down; novel; research study; response; success

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.

描述（由申请人提供）：对敏感和准确方法的需求增加，以测量以多重格式的特定mRNA水平，同时快速且具有成本效益。例如，诸如siRNA之类的基因靶向剂需要监测预期目标的特定敲击以及对其他靶标的意外影响。 theranotics的新兴领域“治疗学和诊断的整合以量身定制患者基因型和仔细监测治疗的进度，尤其需要准确地遵循药物治疗对基因治疗对基因表达的影响，包括区分相关基因。尽管最近几年取得了进展，尽管在几年中取得了进展，但目前在生物学上仍具有基于技术限制的特定rna水平。尽管敏感且合理的准确性，但基于信号放大的另一组方法，每种分析物以及细胞RNA的费力分离，可以避免纯化和复制目标序列，因此在这些步骤中不可能进行跨越的偏置，并且不容易发生这些方法。为了解离非特异性杂种无法确保对AT-和GC富含靶标的无偏见。 我们提出了一种新的方法，用于快速，准确的mRNA定量，具有区分单核苷酸多态性（SNP）和易于多重的能力。该方法结合了经过验证的技术的元素，包括固相杂交和基于珠的多重型，并使用了结合了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拉索）用于特异性地结合和自圆形围绕感兴趣的目标RNA在细胞RNA的样品中，从而形成拓扑连接的复合物，以抵抗我们用来消除背景杂交的严格洗涤条件。然后将套索“解码”，以通过使用标准多路复用程序来量化与感兴趣的各种目标互补的序列的存在。该方法不需要目标扩增，但允许信号放大。该技术可用于对任何感兴趣的基因的高通量分析。作为第一个应用，我们计划开发用于在临床样品中同时定量和基因分型的测定法，以帮助确定适当的治疗方案并遵循治疗进展。该系统对于目前正在开发多种疾病（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的能力很重要。