Quantitative high-throughput nucleic acid assays on a sequencing chip

描述：从基因表达到表观遗传控制，核酸-蛋白相互作用是多种生物过程的基础。虽然DNA或RNA的初级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（尤其是RNA）的组合复杂性，要想全面了解多重序列扰动的影响，就必须采用高通量的方法来分析核酸物种。为此，我们开发了一个平台，可以在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分析。通过生成待探测的不同DNA序列文库，我们构建了一个事后DNA阵列，使用测序数据来定义芯片上克隆簇（每个包含约500个DNA片段）的序列。为了探测RNA结构，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，单链RNA通过稳定的、停滞的RNAP与起源DNA结合。利用这种RNA阵列和定制的荧光分析软件，我们展示了对荧光标记的MS2外壳蛋白（一种典型的RNA结合蛋白）结合亲和力的全面研究。通过测量一致茎环序列中所有可能的单点、双点和三点突变体的平衡常数和MS2的脱轨率，我们证明了这种综合分析在理解相互作用的晶体结构背景下的结构-功能关系以及理解这些相互作用的进化功能约束的能力。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来测量芯片上的RNA结构，包括基于fret的方法来实现热力学熔化测量。利用这些方法，我们将在序列空间中进行RNA稳定性的大量测量，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，我们将观察不同DNA发夹的折叠动力学，为数百万种不同核酸结构的单分子方法打开大门。

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.