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: 8927042
• 负责人: William James Greenleaf
• 金额: $ 30.19万
• 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/8927042
• 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; Health; 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; Signal Transduction; Specificity; Structure; Structure-Activity Relationship; Temperature; Thermodynamics; Time; Variant; base; biophysical properties; combinatorial; computerized data processing; ds-DNA; fluorescence imaging; genome-wide; human disease; instrument; instrumentation; melting; mutant; nucleic acid structure; single molecule; single-molecule FRET; stem; targeted sequencing; temporal measurement; tool; transcriptome sequencing

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过程的基础。虽然DNA或RNA的初级序列决定了建立核酸生物功能的结构图景，但我们预测序列中的扰动如何影响这种结构-功能关系的能力是有限的，即在分子内或分子间相互作用水平上。由于这些核酸聚合物--尤其是RNA--的组合复杂性，要想全面了解多个程度的序列扰动的影响，必然需要高通量的核酸物种分析方法。为此，我们开发了一个在Illumina测序芯片上对数千万到数亿种不同的DNA或RNA分子进行定量生物化学的平台。通过生成一个不同的待探测DNA序列文库，我们已经构建了一个后自组织DNA阵列，使用测序数据来定义芯片上克隆簇的序列(每个克隆簇包含大约500个DNA片段)。为了探测RNA结构，其中最需要组合研究来探测结构和功能，我们使用大肠杆菌RNA聚合酶将固定的dsDNA片段转录成单链RNA，该RNA通过稳定、停滞的RNAP与其起源的DNA结合。利用这个RNA阵列和定制的荧光分析软件，我们已经全面地研究了荧光标记的MS2外壳蛋白的结合亲和力，MS2外壳蛋白是一种典型的RNA结合蛋白。通过测量一致的茎环序列所有可能的单、双和三点突变体的MS2的平衡常数和失效率，我们展示了这种综合分析在相互作用的晶体结构背景下理解结构-功能关系以及理解这些相互作用的进化功能限制的力量。通过开发三种不同的方法来生成芯片上不同的DNA和RNA文库，我们将探索Cas9和TALEN在所有近同源序列和整个基因组中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的特异性以及它们与靶外结合的倾向的详细生物物理信息。我们还将开发三种用于测量芯片上RNA结构的正交方法，包括基于FRET的方法，以实现热力学熔化测量。使用这些方法，我们将在整个序列空间进行大规模的RNA稳定性测量，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热力学测量的数量增加许多数量级，并将很容易地添加到当前的RNA结构预测套件中。最后，我们将把这个高通量平台的灵敏度提升到单分子水平。作为原理证明，我们将观察潜水员DNA发夹折叠的动力学，打开跨越数百万不同核酸结构的单分子方法的大门。