Download Molecular characterization of individual DNA double strand breaks

CODECS 2013 Workshop. San Lorenzo de El Escorial, Madrid, 18th –22nd April, 2013
Molecular characterization of individual DNA double strand breaks
with Tip Enhanced Raman Scattering (TERS) supported by QM/MM
model
Ewelina Lipieca, Jakub Bieleckib, Bayden R. Woodc, Wojciech M. Kwiateka
a
The Henryk Niewodniczanski Institute of Nuclear Physics, PAN, 31-342 Kraków, Poland
Swierk Computing Centre Project, National Centre for Nuclear Research, Sołtana 7, 05400 Otwock-Swierk, Poland
b
c
Centre for Biospectroscopy, School of Chemistry, Monash University, 3800, Victoria,
Australia;
DNA double strand breaks (DSBs) are deadly lesions that can lead to genetic defects and cell
apoptosis1. Techniques to directly image DSBs in isolated DNA include scanning electron
microscopy2, Atomic Force Microscopy (AFM) and single molecule fluorescence microscopy3.
While these techniques can be used to identify DSBs they provide no information on the
molecular events occurring at the break. Tip Enhancement Raman Scattering (TERS) can provide
molecular information from DNA at the nano-scale and in combination with AFM provides a
new way to visualize and characterize DSBs. In experimental part of this study pUC18 plasmid
DNA was fixed onto mica surface and the susceptibility of various kinds of DNA backbone
bonds to UV-C radiation was investigated. Obtained results were compared with calculated
spectra. Computations of Raman activity for untreated and radiation-induce damaged (double
strand break within O-C bond region) DNA structures have been carried out using GAUSSIAN
09 software package in the framework of hybrid QM/MM method (ONIOM model) with Density
Functional Theory formalism with B3LYP hybrid exchange-correlation functional using 6311++G(d,p) basis functions set for a guanine-cytosine pair and Universal Force Field model for
the rest of the system (additional 6 pairs). Initial positions of atoms were taken form Nucleic
Acid Database. Phosphate groups atoms positions were fixed during optimization due to
interaction with divalet cations Mg2+ present on the mica surface. Then, Raman activities were
calculated basing on the optimized geometries.
The appearance of P-O-H motions and CH2, CH3 bending and wagging modes are observable in
experimental and calculated spectra of damaged DNA, what confirmed that broken DNA
fragments are cleaved at the 5’- bonds upon exposure to UV-C radiation.
References
1. M. Norval, Photochem. Photobiol. Sci. 10, 199-225 (2011).
2. M. Brezeanu J. Biol. Phys. 35, 163-174 (2009).
3. E. M. Filippova, Biophys. J. 84, 1281–1290 (2003).