ABSTRACT
Advanced impedance spectroscopy analysis based on the transmission line model (TLM) is explored as a novel QCM acoustic biosensing platform for the detection of the single point mutation effect on the binding of the transcription factors (TFs) to immobilized DNA oligoduplexes and the characterization of the protein-DNA mechanical properties.
Subject(s)
Dielectric Spectroscopy/methods , Immobilized Nucleic Acids/metabolism , Point Mutation , Quartz Crystal Microbalance Techniques/methods , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Immobilized Nucleic Acids/chemistry , Models, Molecular , Protein Binding , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Transcription Factors/chemistryABSTRACT
A hydrogenated amorphous silicon (a-Si:H) photosensor was explored for the quantitative detection of a HIV-1 virion infectivity factor (Vif) at a detection limit in the single nanomolar range. The a-Si:H photosensor was coupled with a microfluidic channel that was functionalized with a recombinant single chain variable fragment antibody. The biosensor selectively recognizes HIV-1 Vif from human cell extracts.
Subject(s)
HIV Infections/diagnosis , HIV Infections/virology , HIV-1/isolation & purification , Microfluidic Analytical Techniques/instrumentation , Silicon/chemistry , vif Gene Products, Human Immunodeficiency Virus/isolation & purification , HEK293 Cells , Humans , Optics and Photonics/instrumentationABSTRACT
Quartz Crystal Microbalance (QCM) biosensor technology was used to study the interaction of the DNA-binding domain (DBD) of the transcription factor RXRα with immobilized specific (DR1) and unspecific (DR1neg) DNA oligoduplexes. We identify the QCM sensor frequency at the susceptance minimum (fBmin) as a better measuring parameter, and we show that fBmin is proportional to the mass adsorbed at the sensor surface and is not influenced by interferences coming from viscoelastic variations of the adsorbed layers or buffers. This parameter was used to study the binding of RXRα to DNA and to calculate the association and dissociation kinetic constants of RXRαDBD-DR1 interaction. We show that RXRαDBD binds to DNA both as a monomer and as a homodimer, and that the mechanism of binding is salt dependent and occurs in two steps. The QCM biosensor data reveal that a high ionic strength buffer prevents the unspecific interactions and at a lower ionic strength the dissociation of RXRαDBD-DR1 occurs in two phases.
Subject(s)
DNA/metabolism , Retinoid X Receptor alpha/metabolism , Base Sequence , DNA/chemistry , Humans , Kinetics , Protein Binding , Protein Interaction Domains and Motifs , Quartz Crystal Microbalance Techniques , Retinoid X Receptor alpha/chemistryABSTRACT
A novel quartz crystal microbalance (QCM) analytical method is developed based on the transmission line model (TLM) algorithm to analyze the binding of transcription factors (TFs) to immobilized DNA oligoduplexes. The method is used to characterize the mechanical properties of biological films through the estimation of the film dynamic shear moduli, G and G, and the film thickness. Using the Saccharomyces cerevisiae transcription factor Haa1 (Haa1DBD) as a biological model two sensors were prepared by immobilizing DNA oligoduplexes, one containing the Haa1 recognition element (HRE(wt)) and another with a random sequence (HRE(neg)) used as a negative control. The immobilization of DNA oligoduplexes was followed in real time and we show that DNA strands initially adsorb with low or non-tilting, laying flat close to the surface, which then lift-off the surface leading to final film tilting angles of 62.9° and 46.7° for HRE(wt) and HRE(neg), respectively. Furthermore we show that the binding of Haa1DBD to HRE(wt) leads to a more ordered and compact film, and forces a 31.7° bending of the immobilized HRE(wt) oligoduplex. This work demonstrates the suitability of the QCM to monitor the specific binding of TFs to immobilized DNA sequences and provides an analytical methodology to study protein-DNA biophysics and kinetics.
