Kinetic Studies of Sequence-Specific Binding of GCN4-bZIP Peptides to DNA Strands Immobilized on a 27-MHz Quartz-Crystal Microbalance

Abstract

Specific protein-DNA interaction was studied quantitatively by using a highly sensitive 27-MHz quartz-crystal microbalance (QCM). Biotinylated DNA double strands (21 bp, having a CRE site of 5'ATGACGTCAT3') were immobilized on an avidin-bound QCM surface, and sequence-specific binding of bZIP 56-mer peptides (having both the basic region for binding and the leucine zipper region for dimerization) to the DNA strand on the QCM was observed. The binding amount (Deltam) at the nanogram level and kinetic parameters such as association constants (Ka) and binding and dissociation rate constants (k1 and k-1) could be obtained from time courses of QCM frequency decreases. A bZIP peptide as a dimer was observed to bind sequence-specifically to one DNA strand having a CRE site. Ka values of ss-bZIP, in which the leucine-zipper region of bZIP was substituted by a Cys-Cys linkage, were largely decreased, and the sequence selectivity also disappeared. Ka values obtained by the QCM method showed good agreement with those obtained from the conventional gel mobility shift assay or from circular dichroism spectrum changes. When the specific sequence of the CRE site of DNA strands was partly changed, Ka values decreased by about a half due to the increase of the dissociation rate constant (k-1) independent of the binding rate constant (k1).

A Novel Tool for Detecting Amyloid Deposits in Systemic Amyloidosis In Vitro and In Vivo

“…Figure 3 indicates the affinity of BSB for TTR, FAP amyloid fibrils, or DRA amyloid fibrils. QCM is a new tool for measuring the affinity of a protein for a protein, a protein for a DNA, and/or a protein for a chemical compound (Matsuno et al, 2001;Okahata et al, 1998). Our results confirmed the strong affinity of BSB for amyloid fibrils but not for the amyloidogenic protein.…”

“…DRA amyloid fibrils were purified from an intestinal sample from a patient as described by Naiki et al (1989). The affinity of BSB or Congo red for amyloid fibrils was examined by using a highly sensitive 27-MHz QCM (Affinix Q; Iitium Company, Tokyo, Japan) as described (Matsuno et al, 2001;Okahata et al, 1998). Briefly, 2 l of 100 g/ml purified amyloid fibrils, 2 l of 100 g/ml purified wild-type TTR, or the variant TTR (ATTR Val30Met) was directly immobilized on a QCM plate, the plate was soaked in 6 ml of a buffered solution [10 mM Tris-HCl, 10% dimethyl sulfoxide (DMSO), pH 7.4] at 25°C, and the resonance frequency of the QCM was defined as the 0 position after equilibrium.…”

“…To determine the usefulness of the compound in vivo, we also examined BSB reactivity in amyloid deposits in mice in which AA amyloidosis had been induced. In addition, we investigated the affinity of BSB for amyloid fibrils and the inhibitory effect of BSB on formation of transthyretin (TTR) amyloid fibrils in vitro by means of a highly sensitive 27-MHz quartz crystal microbalance (QCM) (Matsuno et al, 2001;Okahata et al, 1998) and electron microscopy, respectively. …”

A Highly Sensitive 27 MHz Quartz-Crystal Microbalance as a Device for Kinetic Measurements of Molecular Recognition on DNA Strands

“…The 27 MHz QCM is highly sensitive and quantitative enough to detect various molecular recognitions on DNA molecules, such as DNA-DNA hybridization, 41 sequence specific binding of bZIP peptide to dsDNA, 42 and a series of DNA polymerase reactions such as the binding process of enzyme, the elongation rate and the release of the enzyme from the completely polymerized DNA). 43 Since the QCM is a mass-measuring device, we can apply it widely to detect various host-guest interactions: adsorption of bitter or odor substances to lipid membrane, 22 peptide-DNA interactions, 44 intercalation of antibiotics to DNA strands, and in vitro selection of DNA binding to host molecules.…”

Ferrocenylnaphthalene Diimide-based Electrochemical Ribonuclease Assay

“…9 After the electrodes were dried by nitrogen gas, they were soaked in a 1 mM 3,3′-dithiodipropionic acid aqueous solution for 2 h at room temperature. 22 After the electrodes were washed with Gengard water, 30 μl of a solution containing 5 mM EDC and 8 mM NHS in 0.02 M phosphate buffer (pH 7.0) was placed on a gold electrode held upside down and kept in a clean bench for 20 min. After the electrodes were washed with Gengard water, 2.5 -10 ng/μl of poly(A) + RNA from mouse kidney was placed on the electrode for 20 min at room temperature.…”