Quantitative high-throughput nucleic acid assays on a sequencing chip

测序芯片上的定量高通量核酸测定

• 批准号: 8766567
• 负责人: William James Greenleaf
• 金额: $ 29.41万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8766567
• 关键词: Accounting; Acute; Address; Affect; Affinity; Base Sequence; Binding; Biochemistry; Biological; Biological Assay; Biological Process; Biology; Capsid Proteins; Cells; ChIP-seq; Communities; Computer software; Consensus; Custom; DNA; DNA Folding; DNA Library; DNA Sequence; DNA-Binding Proteins; DNA-Directed RNA Polymerase; DNA-Protein Interaction; Data; Data Analyses; Data Set; Data Sources; Defect; Disease; Ensure; Enterobacteria phage MS2; Enzymes; Epigenetic Process; Equilibrium; Escherichia coli; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Gene Expression Profile; Genetic Polymorphism; Genome; Heterogeneity; High-Throughput Nucleotide Sequencing; Human Genome; Individual; Investigation; Kinetics; Label; Length; Libraries; Link; MS2 coat protein; Massive Parallel Sequencing; Measurement; Measures; Methods; Modeling; Molecular; Nucleic Acid Folding; Nucleic Acids; Nucleic acid sequencing; Oligonucleotides; Phenotype; Polymers; Processed Genes; Proteins; Protocols documentation; RNA; RNA Probes; RNA Sequences; RNA Stability; RNA-Binding Proteins; RNA-Protein Interaction; Relative (related person); Research; Research Infrastructure; Resolution; Signal Transduction; Specificity; Structure; Structure-Activity Relationship; Temperature; Thermodynamics; Time; Variant; base; biophysical properties; combinatorial; computerized data processing; fluorescence imaging; genome-wide; human disease; instrument; instrumentation; melting; mutant; nucleic acid structure; public health relevance; single molecule; single-molecule FRET; stem; tool

DESCRIPTION: Nucleic-acid-protein interactions are fundamental to diverse biological processes from gene expression to epigenetic control. While the primary sequence of DNA or RNA sets the structural landscape that establishes the biological function of nucleic acids, our ability to predict how perturbations in sequence affect this structure- function relationship eithe at the intra- or inter-molecular interaction level, is limited. Because of the combinatorial complexity of these nucleic acid polymers - especially RNA - obtaining a comprehensive picture of the effects of multiple degrees of sequence perturbation necessarily requires high-throughput methods of assaying nucleic acid species. To this end, we have developed a platform for quantitative biochemistry of tens to hundreds of millions of diverse DNA or RNA molecules on an Illumina sequencing chip. By generating a diverse library of DNA sequences to be probed, we have constructed a post hoc DNA array, using the sequencing data to define the sequences of the clonal clusters (each containing approximately 500 fragments of DNA) on the chip. To probe RNA structures, where the need for combinatorial investigations to probe both structure and function is most acute, we use E. coli RNA polymerase to transcribe the immobilized dsDNA fragments into single stranded RNA, which remains bound to its DNA of origin via a stable, stalled RNAP. Using this RNA array, and custom built fluorescence analysis software, we have demonstrated comprehensive investigations of binding affinities of fluorescently labeled MS2 coat protein, a canonical RNA binding protein. By measuring the equilibrium constants and off-rates for MS2 for all possible single, double, and triple point mutants of the consensus stem-loop sequence, we demonstrate the power of this comprehensive analysis for understanding structure-function relationships in the context of the crystal structure of the interactions, as well as understanding the evolutionary functional constraints of these interactions. By developing three different methods of generating diverse libraries of DNA and RNA on-chip, we will probe the relative affinities of Cas9 and TALEN for target sequences across all near-cognate sequences and across the entire genome. These quantitative investigations will provide detailed biophysical information about the specificity of these protein, as well as their propensity for off-target binding. We will also develop three orthogonal methods for measuring RNA structure on-chip, including FRET-based methods to enable thermodynamic melting measurements. With these methods, we will carry out massive measurements of RNA stability across sequence space, probing all possible short hairpin structures as well as internally mismatched stem loops. These data will multiply the number of thermodynamic measurements of RNA by many orders of magnitude, and will be easily added to current RNA structure prediction suites. Finally we will push the sensitivity of this high-throughput platform o the single molecule level. As proof-of-principle, we will observe the kinetics of folding of divers DNA hairpins, opening the door to single-molecule methods across millions of diverse nucleic acid structures.

描述：核酸 - 蛋白质相互作用是从基因表达到表观遗传控制的各种生物学过程的基础。虽然DNA或RNA的主要序列设置了建立核酸生物学功能的结构景观，但我们预测序列扰动如何影响这种结构 - 功能关系的能力受到限制。由于这些核酸聚合物（尤其是RNA）的组合复杂性，因此获得了多个序列扰动的效果的全面图片，必须需要高通量的方法来分析核酸物种。为此，我们开发了一个平台，用于在光照射测序芯片上进行数十至数亿种不同的DNA或RNA分子的平台。通过生成要探测的DNA序列的多元化库，我们使用测序数据构建了一个事后DNA阵列，以定义芯片上克隆簇的序列（每个都包含大约500个DNA片段）。为了探测RNA结构，在探测结构和功能的组合研究的需求最为急性的情况下，我们使用大肠杆菌RNA聚合酶将固定的DSDNA片段转录到单个绞合的RNA中，该片段始终通过稳定的，停滞的RNAP绑定到其原始的DNA。使用此RNA阵列和自定义构建的荧光分析软件，我们已经证明了对荧光标记的MS2涂层蛋白的结合亲密关系的全面研究，MS2涂层蛋白是一种规范的RNA结合蛋白。通过测量所有可能的单个，双重和三点突变体的平衡常数和离速率的平衡常数和离数，我们证明了这种综合分析对于理解相互作用晶体结构背景下的结构 - 功能关系的能力，以及理解这些相互作用的进化功能约束。通过开发三种不同的方法来生成片上DNA和RNA的不同库，我们将探测Cas9和Talen的相对亲和力，以探测所有近近似序列以及整个基因组的目标序列的相对亲和力。这些定量研究将提供有关这些蛋白质特异性的详细生物物理信息，以及它们的脱靶结合倾向。我们还将开发三种正交方法，用于测量片上RNA结构，包括基于FRET的方法以实现热力学熔化测量。通过这些方法，我们将对序列空间进行大规模测量RNA稳定性，探测所有可能的短发夹结构以及内部不匹配的茎环。这些数据将使RNA的热力学测量数量乘以许多数量级，并将很容易地添加到当前的RNA结构预测套件中。最后，我们将推动这个高通量平台的灵敏度o单分子水平。作为原则证明，我们将观察潜水DNA发夹折叠的动力学，为数百万种不同核酸结构的单分子方法打开门。