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Thermal Denaturation of DNA studied by Ultrafast 2D-IR Spectroscopy
Gordon R. Hithell1, Daniel J. Shaw1, Lennart Ramakers1, John May3, Gregory M. Greetham2, Michael Towrie2, Anthony W. Parker2, Glenn A. Burley3, Matthew Baker3, Neil T. Hunt1 Department of Physics, University of Strathclyde, SUPA, Glasgow, UK Central Laser Facility, STFC Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot, UK WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, UK Contact Abstract - The thermal denaturation of two types of DNA duplexes has been studied using ultrafast two-dimensional infrared spectroscopy (2D-IR) UV-visible absorption spectroscopy. Using the 1600cm cm-1 region as a signature for infrared red modes sensitive to hydrogen bonding due to Watson-Crick base pairing, 2D-IR will offer ultrafast time resolution to investigate rapid dynamic processes that underpin the denaturation of a DNA duplex. These measurements were supported with data obtained using linear FT-IR. The duplex DNA systems studied were a 10mer d(GGAAATTTGC)2 and an AT rich 15mer d(ATTATTATTATATTA)2. 2D-IR spectra at a range of temperatures between 20 and 80oC were obtained and the changes in the vibrational modes and their couplings were correlated with temperature using Principal Component Analysis (PCA). Comparisons to FT-IR and UV-vis measurements demonstrate that they are attributable to duplex melting. Using this approach, the spectral contributions due to inter-base coupling resulting from Watson-Crick base pairing and how they change upon denaturation have been separated for both AT and GC components of the duplex for the first time. This combination of ultrafast 2D-IR and PCA allows us to separate spectral signatures of AT and GC base pairs and to probe their dynamics on the fs-ps timescales on which hydrogen bonds exchange. This will provide insight into transient processes associated with breaking of inter-base hydrogen bonds and solvation of the DNA macromolecule; providing a complementary picture to that obtained by crystallography and NMR. Introduction Experimental Methods 2D IR uses a train of ultrafast broadband femtosecond pulses to interrogate a sample. Two pump pulses followed by a probe pulse are incident on the sample, following this a signal field is then emitted from the sample and the information is spread over two frequency axes [1]. The ultrashort laser pulses used in this experiment offer extremely high time resolution and the ability to observe dynamic processes that occur on the order of picoseconds. The time resolution available from this technique allows the observation of any molecular fluctuations occurring on these timescales that could play an important role in the melting of the duplex structure that would not be observable using linear spectroscopic methods. DNA is the primary repository of genetic information in almost all living organisms. Despite this central role, the mechanisms that DNA-proteins and small molecules identify their target sequences are still unclear. The first step in the reading of this genetic code by the biological machinery in organisms is the unwinding of the double stranded helix into single strands in order for the DNA base sequence to be read. This is often modelled by thermally unfolding the duplex to simulate this effect. Fig. 1 – DNA double helix highlighting major and minor groove formation. Tw pump Sample Detector probe τ The aim of this work is to begin to understand how different base-pairs in various contexts influence denaturation of a duplex structure and the manner in which the duplex melts. There are several models proposed for melting such as peeling or random dissociation, however the time scales that hydrogen bonds break are beyond the time resolution of NMR and UV. Ultrafast 2D-IR spectroscopy shows high sensitivity to changes in hydrogen bonding conformations and has a temporal resolution fast enough to capture these fluctuations which will provide insight into the dynamic process of denaturation. In this work, UV-Visible, FTIR and 2D-IR spectroscopies have been employed to study the melting of two DNA duplexes, the first a known target sequence for minor groove binder molecules and also an A-T rich sequence to highlight the spectral contributions of the A-T portion of the mixed sequence Fig. 3 – Typical experimental set up of a 2D-IR measurement showing pulse sequences Ultrafast 2D-IR methods offer a dynamic perspective on the duplex melting process which is traditionally characterised by UV-visible spectroscopy. Once melting profiles for the selected DNA sequences have been established using linear spectroscopic methods, 2D-IR can provide further information on the coupling between vibrational modes of DNA bases, changes in the off-diagonal coupling patterns could provide information on the melting pathway such as A-T base pairs breaking before G-C pairs and forming bubbles in the duplex. Fig. 2 – DNA base pairs Linear Spectroscopy Fig. 6– FTIR Spectra of our AT 15mer DNA duplex recorded between 20oC (blue) and 80oC (red). Similar changes in the FTIR spectrum to those seen in the 10mer sequence are observed. Fig. 4 – Normalised UV-Vis melting curves for 10mer and AT 15mer DNA duplexes. Melting temperature of the sequences used in the experiments are determines, ~42oC for the 10mer and ~32oC for the AT 15mer. Fig. 5 – FTIR Spectra of our 10mer DNA duplex recorded between 20oC (blue) and 80oC (red). Vibrational responses from DNA bases such as C=O stretches show large sensitivity to duplex melting d(ATTATTATTATATTA)2 d(GGAAATTTGC)2 UV-Vis 2D-IR Spectroscopy Principal Component Analysis Assignments G (G-C) C (G-C) AT G C d(GGAAATTTGC)2 Fig. 7 – Low and high temperature 2D-IR spectra for 10mer duplex with accompanying linear FTIR spectra. Fig. 8 – (Left) Coefficients of Principal Components (PC) for 10mer comparing FTIR and 2D-IR spectral analysis. (Right) Extracted Principal Component 2 (PC2) spectrum for the 10mer sequence, PC2 indicates features that change as a function of temperature. Fig. 9 – Assignment of low temperature 2D spectrum using GC duplex measurement [2] and high temperature denatured 2D spectrum using 2D of individual DNA bases [4]. AT A (A-T) T (A-T) d(ATTATTATTATATTA)2 Fig. 10 – (Left) Low and high temperature 2D-IR spectra for 10mer duplex with accompanying linear FTIR spectra. Fig. 11 – (Left) Coefficients of Principal Components (PC) for AT 15mer comparing FTIR and 2D-IR spectral analysis. (Right) Extracted Principal Component 2 spectrum for the AT 15mer sequence. Fig. 12 – Assignment of low temperature 2D spectrum using GC duplex measurement [3] and high temperature denatured 2D spectrum using 2D of individual DNA bases [4]. Conclusions For the first time the 2D spectrum of a DNA duplex containing only AT base pairs has been measured experimentally in this region. GC and AT base pair contributions from the complex 2D spectrum are now able to be separated and will now allow us to begin to make assignments of the off diagonal features of the 2D spectrum. Changes in off-diagonal coupling patterns will provide new insight into the dynamic hydrogen bonding processes that govern denaturation of the DNA duplex not available from UV or NMR spectroscopies. References Hunt, N. T. Chem.Soc.Rev., 38, 2009 Krummel, A., Zanni, M., J. Phys. Chem. B, Vol. 110, No. 28, 2006 Lee, C. Cho, M., J. Chem. Phys. 126, 2007 Peng, C. Jones K. Tokmakoff A. J. Am. Chem. Soc. 133 , 2011
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