Issue 8, 2014

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, Gfilm and G′′film, 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 (HREwt) and another with a random sequence (HREneg) 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 HREwt and HREneg, respectively. Furthermore we show that the binding of Haa1DBD to HREwt leads to a more ordered and compact film, and forces a 31.7° bending of the immobilized HREwt 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.

Graphical abstract: Conformational and mechanical changes of DNA upon transcription factor binding detected by a QCM and transmission line model

Supplementary files

Article information

Article type
Paper
Submitted
05 Sep 2013
Accepted
30 Oct 2013
First published
31 Oct 2013

Analyst, 2014,139, 1847-1855

Conformational and mechanical changes of DNA upon transcription factor binding detected by a QCM and transmission line model

J. de-Carvalho, R. M. M. Rodrigues, B. Tomé, S. F. Henriques, N. P. Mira, I. Sá-Correia and G. N. M. Ferreira, Analyst, 2014, 139, 1847 DOI: 10.1039/C3AN01682J

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