21.2: Kinetic Studies of Molecular Recognition on DNA by using a 27 MHz QCM

摘要

DNA-binding proteins play a key role in fundamental cellular processes such as transcription for mRNA synthesis and DNA replication. Understanding an interaction of DNA-binding protein is important to study these biological processes on a molecular level and to design a new DNA-binding protein/peptide that recognizes any DNA sequence to control any gene expression. Physical kinetic studies of DNA-protein interactions have been mainly performed by gel mobility shift assay. This technique is widely used in the field of molecular biology; however, it has some difficulties with regard to quantitatively detecting and an observation of the time course on these interactions. In this paper, we introduce a 27-MHz quartz-crystal microbalance (QCM) as a new tool to detect DNA-protein/peptide interactions in an aqueous solution. A QCM is known to provide very sensitive mass measuring devices because its resonance frequency decreases linearly upon the increase of a mass on the QCM electrode at the nanogram level. From the time course of the frequency decrease (mass increase), the binding amount (Δm), association constant (K_(a)), binding and dissociation rate constant (k_(1) and k_(-1)) can be obtained. We undertook observations of a various protein/peptide (for example, histone and transcription factors such as a Zinc-finger type peptide or a Leucine-zipper type peptide) binding onto double strand DNA (dsDNA) included their binding site. When dsDNA was immobilized on a QCM, sequence-specific binding of protein/peptide could be followed as a frequency decrease (mass increase). The 27-MHz QCM is highly sensitive and quantitative enough to detect various molecular interactions such as DNA-binding protein/peptide binding onto dsDNA. Since the QCM is a mass-measuring device, we can apply it widely to detect various interactions of a biomolecular such as DNA, RNA, protein/peptide, and sugar.