Analysis of Multidimensional G‐Quadruplex Melting Curves
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Abstract
Multidimensional ?3D? melting curves for G?quadruplexes are obtained by recording whole spectra (absorbance, CD, fluorescence) as a function of temperature, rather than the common approach of recording the spectral response to temperature at a single wavelength. 3D melting curves are richer in information, and can be used to enumerate the number of significant species and intermediate states required to properly analyze the thermal denaturation reaction to obtain thermodynamic information. This unit describes the application of the method of singular value decomposition to the analysis of 3D melting data obtained for G?quadruplex structures, and how the results of such an analysis can be used to provide a more complete characterization of the mechanism of quadruplex unfolding. Curr. Protoc. Nucleic Acid Chem. 45:17.4.1?17.4.16. © 2011 by John Wiley & Sons, Inc.
Keywords: G?quadruplex; thermodynamics; spectroscopy; thermal melting; singular value decomposition
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Overview of SVD Analysis
- Basic Protocol 1: Analysis of Multidimensional Melting Data
- Commentary
- Literature Cited
- Figures
- Tables
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Figure 17.4.1 Multidimensional melting data and the structure of data sets used for analysis. (A ) Examples of UV spectra as a function of temperature. (B ) Structure of the data matrix constructed from multidimensional melting data. (C ) Columns of the data matrix show the UV spectrum at a single temperature. (D ) Rows of the data matrix show the melting curve at a single wavelength. View Image -
Figure 17.4.2 Flowchart for the analysis of multidimensional melting data. View Image -
Figure 17.4.3 Temperature‐dependence of the UV absorption, CD, and FRET properties of the oligonucleotide d[A(GGGTTA)3 ] (143D), which forms a quadruplex structure in 50 mM NaCl. Panels A , C , and E in the figure show spectroscopic measurements for each method as a function of increasing temperature. The arrows represent the direction of the signal change as the temperature is increased. Panels B , D , and F compare the corresponding first derivative of the signal with respect to temperature at the indicated wavelengths. The points in panels A, C, and E were obtained from the raw data by smoothing with a 9‐point, second order Savitzky‐Golay filter prior to calculation of the first derivative using the program Origin 7.0. Each solid line in the derivative plot represents a nonlinear least‐squares fit of the smoothed data points to a two‐state F ↔ U mechanism described by d S (λ)/d T = A f (1− f ) T 2 . Note that the data points are not well‐represented by the two‐state model. In addition, the apparent mid‐point temperature, as indicated by the number above the maximum or minimum of each curve, depends on both the wavelength of observation, as well as the method of measurement. Both of these factors indicate that the two‐state model is unable to accurately describe the temperature‐dependence of the optical signal. The absorbance and CD measurements were carried out with 143D, and FRET measurements were carried out with 6‐Fam‐143D‐Tamra. Conditions: 10 mM tetrabutyl ammonium phosphate, 50 mM NaCl, 1 mM EDTA, pH 7.0. Oligonucleotide concentrations were: 4.6 µM for the absorbance experiments, 4.7 µM for the CD experiments, and 0.1 µM for the fluorescence experiments. View Image -
Figure 17.4.4 Charts showing the singular values and their relative variance for the first ten significant components of the UV absorbance (A ), CD (B ), and FRET (C ) data in Figure . View Image -
Figure 17.4.5 Basis spectra determined using SVD analysis of the UV absorbance (A ), CD (B ), and FRET (C ) data sets in Figure by MATLAB. The numbers in the plot legends correspond to the rank order of the significant spectroscopic species. View Image -
Figure 17.4.6 Panels A , C , and E show plots of the four or five most significant elements of the V matrices corresponding to the data sets in Figure . Panels B (absorbance data), D (CD data), and F (FRET data) show the autocorrelation coefficients estimated for the first ten significant components of the U and V matrices. View Image -
Figure 17.4.7 Analysis of absorbance, CD, and FRET data matrices of Figure by GlobalWorks. The absorbance and CD data were fit to a sequential four‐state melting process F ↔ I1 ↔ I2 ↔ U, where F and U represent the folded and unfolded states, and I1 and I2 are spectroscopic intermediates. EV = eigenvalue, which for the spectra correspond directly to the measured spectroscopic parameter. For the species distribution plots in Panels B , D , and F , EV is related to the relative contribution of each species to the total signal at the particular temperature. Also shown in panels B, D, and F is a comparison of the experimental data (•) and the best fit (solid line) calculated using the least‐squares optimized parameters in Table . In addition, the distribution of residuals is shown (‐o‐). Panels A , C , and E show the calculated UV, CD, and fluorescence emission spectra, respectively, for the folded, intermediate, and unfolded species. View Image
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Literature Cited
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