title: Conformational and mechanical changes of DNA upon transcription factor binding detected by a QCM and transmission line model.
abstract: 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.
authors.affiliation: IBB-Institute for Biotechnology and Bioengineering, Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, 8005-139 Faro, Portugal. gferrei@ualg.pt.
authors.forename: Jorge
authors.initials: J
authors.lastname: de-Carvalho
authors.forename: Rogério M M
authors.initials: RM
authors.lastname: Rodrigues
authors.forename: Brigitte
authors.initials: B
authors.lastname: Tomé
authors.forename: Sílvia F
authors.initials: SF
authors.lastname: Henriques
authors.forename: Nuno P
authors.initials: NP
authors.lastname: Mira
authors.forename: Isabel
authors.initials: I
authors.lastname: Sá-Correia
authors.forename: Guilherme N M
authors.initials: GN
authors.lastname: Ferreira
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D008962:Models, Theoretical; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011791:Quartz; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: C439059:Haa1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011791:Quartz; D004247:DNA