Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers Lu Sun, † Karolin Frykholm, † Louise H. Fornander, † Sofia Svedhem, ‡ Fredrik Westerlund, † and Bjö rn Åkerman* ,† † Department of Chemical and Biological Engineering and ‡ Department of Applied Physics, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden * S Supporting Information ABSTRACT: Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigat- ing DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oli- gonucleotides of designed base sequences. The DNA films were ex- posed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dis- sociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film deformation allows comparative studies of two or more analytes using the same DNA layer as demonstrated here by spermine and spermidine. ■ INTRODUCTION DNA biosensors utilize an oligonucleotide as a recognition ele- ment for detecting binding of biomolecules, as used in clinical diagnosis, 1 genetic analysis, 2,3 environmental monitoring, 4 and food analysis. 5 DNA biosensors consisting of single-stranded DNA probes immobilized on a transducer surface may be used to recognize their complementary DNA strands via hybrid- ization, 6,7 but also interactions between DNA and ligands, for instance proteins or small molecules such as duanomycin, may be monitored. 8 So far, DNA biosensors have exploited trans- ducer technologies including electrochemical, 1-3 optical, 9 pie- zoelectric, 4 and surface plasmon resonance (SPR) 5,10 methods. These methods have demonstrated considerable success regard- ing rapidness, simplicity, and sensitivity but do not take advan- tage of information regarding structural changes in the DNA upon binding of the target molecules. The quartz crystal microbalance with dissipation monitoring (QCM-D) is a powerful tool for real-time detection of bio- molecule adsorption to solid/liquid interfaces. It provides simu- ltaneous monitoring of changes in mass and viscoelastic pro- perties of an adsorbed layer through detection of shifts in the resonance frequency (Δf) and the damping (energy dissi- pation, ΔD) of the oscillatory motion of the quartz sensor. QCM-D has been demonstrated to be a sensitive and efficient tool for the study of recognition reactions, involving DNA, 11-18 proteins, 19-21 lipid membranes, 22-26 and cells. 27-30 Notably, QCM-D measures added mass, including potential hydration water brought to or removed from the surface by the adsorbing moleculeand changes in water content of the DNA layers can Received: July 6, 2014 Revised: August 24, 2014 Published: September 8, 2014 Article pubs.acs.org/JPCB © 2014 American Chemical Society 11895 dx.doi.org/10.1021/jp506733w | J. Phys. Chem. B 2014, 118, 11895-11904