Universal, Compact Combinatorial Microarrays for DNA Binding Site Discovery

描述（由申请人提供）：转录因子（TF）及其DNA结合位点之间的相互作用是细胞内调控网络的组成部分。这些相互作用控制着发育和对环境压力反应的关键步骤，在人类中，它们的功能障碍可能导致各种疾病的进展。到目前为止，只有一小部分序列特异性转录因子的特征足以让我们知道它们可以结合的所有序列，同样重要的是，不能结合。该结合位点序列数据的这种稀疏性是高度成问题的，因为这些稀疏数据集随后用于搜索这些位点的基因组出现，其中预测了许多假阳性和假阴性结合位点。最终，生物界需要的是关于所有可能的DNA序列变体的更完整的TF结合位点数据。这些数据将使我们能够提高准确性，我们可以预测基因组序列内的功能顺式调控元件。我们已经计算出了我们认为是所有可能的结合位点的最大紧凑表示，仍然允许DNA结合分子的序列特异性被恢复。该技术的优点是所有可能的DNA序列变体都可以以空间和成本有效的方式在DNA微阵列上表示，使得仅需要合成最小数量的单个DNA序列和单个DNA点。在这个项目中，我们将：（1）开发紧凑组合DNA微阵列在蛋白质结合微阵列（PBM）实验中的用途，用于鉴定序列特异性TF的所有可能的DNA结合位点;（2）使用紧凑组合DNA微阵列确定所有可能的DNA结合位点对酿酒酵母TF的结合亲和力，并创建这些数据的数据库;和（3）评价完整的结合特异性数据和结合亲和力数据对于改进体内TF结合位点预测的效用。不存在用于确定TF的所有候选DNA结合位点的相对结合亲和力的其它技术，其与紧凑组合DNA微阵列PBM技术一样高通量。这些研究应允许更好地了解TF结合位点在真核基因组中的结合亲和力的重要性。这样的数据也可以增加的准确性与顺式调控模块可以预测在高等真核生物基因组。

DESCRIPTION (provided by applicant): The interactions between transcription factors (TFs) and their DNA binding sites are an integral part of the regulatory networks within cells. These interactions control critical steps in development and responses to environmental stresses, and in humans their dysfunction can contribute to the progression of various diseases. Thus far only a small handful of sequence-specific TFs have been characterized well enough for us to know all the sequences that they can, and just as importantly, can not bind. This sparseness of this binding site sequence data is highly problematic, because these sparse datasets are then used to search for genomic occurrences of these sites, with many false positive and false negative binding sites being predicted. Ultimately what the biological community needs is much more complete TF binding site data on all possible DNA sequence variants. These data will allow us to improve the accuracy with which we can predict functional cis regulatory elements within genomic sequence. We have calculated what we believe is a maximally compact representation of all possible binding sites that still allows the sequence specificities of DNA binding molecules to be recovered. The advantage of this technology is that all possible DNA sequence variants can be represented on DNA microarrays in a space- and cost- efficient manner, so that only a minimal number of individual DNA sequences and individual DNA spots need to be synthesized. In this project, we will: (1) develop the use of compact combinatorial DNA microarrays in protein binding microarray (PBM) experiments for identifying all possible DNA binding sites of sequence-specific TFs; (2) determine the binding affinities of all possible DNA binding sites for -15 Saccharomyces cerevisiae TFs using compact combinatorial DNA microarrays and create a database of these data; and (3) evaluate the utility of complete binding specificity data and binding affinity data for improved prediction of in vivo TF binding sites. There exists no other technology for the determination of the relative binding affinities of all candidate DNA binding sites for TFs that is as high-throughput as the compact combinatorial DNA microarray PBM technology. These studies should permit a better understanding of the importance of the binding affinities of TF binding sites in eukaryotic genomes. Such data may also increase the accuracy with which cis regulatory modules can be predicted in higher eukaryotic genomes.