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: 9336944
• 负责人: William James Greenleaf
• 金额: $ 30.13万
• 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/9336944
• 关键词: Accounting; Acute; Address; Affect; Affinity; Base Sequence; Binding; Biochemistry; Biological; Biological Assay; Biological Process; Biology; Biophysics; Capsid Proteins; Cells; ChIP-seq; Communities; Computer software; Consensus; Crystallization; 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 Process; Genetic Polymorphism; Heterogeneity; High-Throughput Nucleotide Sequencing; Human Genome; Immobilization; Individual; Investigation; Kinetics; Label; Length; Libraries; Link; Liquid substance; MS2 coat protein; Massive Parallel Sequencing; Measurement; Measures; Methods; Modeling; Molecular; Nucleic Acid Folding; Nucleic Acids; Oligonucleotides; Phenotype; Polymers; Proteins; Protocols documentation; RNA; RNA Probes; RNA Sequences; RNA Stability; RNA library; RNA-Binding Proteins; RNA-Protein Interaction; Research; Research Infrastructure; Signal Transduction; Specificity; Structure; Structure-Activity Relationship; Temperature; Thermodynamics; Time; Variant; base; biophysical properties; combinatorial; computerized data processing; ds-DNA; exhaustion; fluorescence imaging; genome-wide; human disease; instrument; instrumentation; melting; mutant; nucleic acid structure; programs; public health relevance; sequencing platform; single molecule; single-molecule FRET; stem; temporal measurement; tool; transcriptome; whole genome

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的一级序列设定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在分子内或分子间相互作用水平上影响这种结构-功能关系的能力是有限的。由于这些核酸聚合物（特别是RNA）的组合复杂性，获得多个程度的序列扰动的影响的全面图片必然需要测定核酸种类的高通量方法。为此，我们开发了一个平台，用于在Illumina测序芯片上对数千万至数亿个不同的DNA或RNA分子进行定量生物化学分析。通过生成待探测的DNA序列的多样化文库，我们构建了一个事后DNA阵列，使用测序数据来定义芯片上的克隆簇（每个克隆簇包含大约500个DNA片段）的序列。为了探测RNA结构，在需要组合研究以探测结构和功能的地方，我们使用E。coli RNA聚合酶将固定的dsDNA片段转录成单链RNA，单链RNA通过稳定的停滞的RNAP与其来源的DNA保持结合。使用这种RNA阵列和定制的荧光分析软件，我们已经证明了荧光标记的MS 2外壳蛋白，一个典型的RNA结合蛋白的结合亲和力的全面调查。通过测量MS 2的所有可能的单，双，和三相点突变体的共识茎环序列的平衡常数和解离速率，我们证明了这种全面的分析理解的晶体结构的相互作用的背景下的结构-功能关系的力量，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在芯片上生成不同的DNA和RNA文库，我们将在所有近同源序列和整个基因组中探测Cas9和TALEN对靶序列的相对亲和力。这些定量研究将提供有关这些蛋白质的特异性以及其脱靶结合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，以实现热力学熔解测量。通过这些方法，我们将在整个序列空间中进行大量的RNA稳定性测量，探测所有可能的短发夹结构以及内部错配的茎环。这些数据将使RNA的热力学测量的数量增加许多数量级，并且将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高到单分子水平。作为原理证明，我们将观察多种DNA发夹折叠的动力学，为跨越数百万种不同核酸结构的单分子方法打开大门。