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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Quantifying DNA-protein binding specificities by using oligonucleotide mass tags and mass spectroscopy
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Quantifying DNA-protein binding specificities by using oligonucleotide mass tags and mass spectroscopy

机译：通过使用寡核苷酸质谱标签和质谱定量DNA-蛋白质结合特异性

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摘要

The ability to determine the relative binding affinity of different transcription-factors (TF) to their DNA binding sites is fundamentally important for a comprehensive understanding of gene regulation. Here we present a general approach for multiplex quantification of DNA-TF binding specificities in vitro using oligonucleotide mass tag (OMT) labeling and mass spertroscopic quantification. An OMT is a short nucleic acid sequence with a distinct mass that can be resolved by a mass spectrometer. Each putative binding sequence is labeled with a unique OMT, and PCR amplification of OMTs is performed after removing nonbound DNA. Subsequently, a primer extension reaction is carried out, and the extension products are quantified by MALDI-TOF mass spectroscopy. Using the TF NF-κB P50, we have quantified the binding specificities of up to 15 binding sequences in a single assay. The results from the multiplex assay are consistent with data from the traditional gel shift assay. The approach allows the competitive binding of multiple DNA sequences to the given protein in a homogeneous reaction. By using the commercially available homogeneous MassEXTEND platform (SEQUENOM), it is scalable for high-throughput DNA-TF binding applications, including genome-wide TF binding site mapping and analyses of SNPs in promoter regions.
机译：确定不同转录因子（TF）与其DNA结合位点的相对结合亲和力的能力对于全面了解基因调控至关重要。在这里，我们介绍了一种使用寡核苷酸质量标签（OMT）标记和质量精镜定量在体外对DNA-TF结合特异性进行多重量化的通用方法。 OMT是具有独特质量的短核酸序列，可以通过质谱仪解析。每个推定的结合序列都用独特的OMT标记，并在去除未结合的DNA后进行OMT的PCR扩增。随后，进行引物延伸反应，并通过MALDI-TOF质谱对延伸产物进行定量。使用TFNF-κBP50，我们在一次测定中定量了多达15个结合序列的结合特异性。多重分析的结果与传统凝胶位移分析的数据一致。该方法允许在均匀反应中将多个DNA序列竞争性结合至给定的蛋白质。通过使用市售的同质MassEXTEND平台（SEQUENOM），它可扩展用于高通量DNA-TF结合应用，包括全基因组TF结合位点作图和启动子区域SNP分析。

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