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Label-free, atomic force microscopy-based mapping of DNA intrinsic curvature for the nanoscale comparative analysis of bent duplexes.

Buzio R, Repetto L, Giacopelli F, Ravazzolo R, Valbusa U - Nucleic Acids Res. (2012)

Bottom Line: We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature.Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far.We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes.

View Article: PubMed Central - PubMed

Affiliation: S.C. Nanobiotecnologie, National Institute for Cancer Research IST, Genova, Italy.

ABSTRACT
We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes.

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(a) Representative conformations of six chains generated by MC methods from the intrinsic 2D trajectory predicted by De Santis et al model for the target DNA. A randomly flat substrate has been intentionally added to generate topographies resembling as close as possible those obtained by AFM (b) Theoretical pattern of variation of the CP for two different values of sliding windows length L; encircled are the main peaks of the plots. Experimental results are reported for comparison with L = 34 nm. (c) Intrinsic curvature of the 2D trajectory predicted by De Santis et al model, with marked positions of the pairs of segments of length L = 17 nm (50 bp) related to the peaks highlighted in (b). Positions are shown for clarity also on the 2D chain. (d) Comparison of experimental results with the theoretical pattern of variation of the CP predicted with the model of Bolshoy et al.
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gks210-F6: (a) Representative conformations of six chains generated by MC methods from the intrinsic 2D trajectory predicted by De Santis et al model for the target DNA. A randomly flat substrate has been intentionally added to generate topographies resembling as close as possible those obtained by AFM (b) Theoretical pattern of variation of the CP for two different values of sliding windows length L; encircled are the main peaks of the plots. Experimental results are reported for comparison with L = 34 nm. (c) Intrinsic curvature of the 2D trajectory predicted by De Santis et al model, with marked positions of the pairs of segments of length L = 17 nm (50 bp) related to the peaks highlighted in (b). Positions are shown for clarity also on the 2D chain. (d) Comparison of experimental results with the theoretical pattern of variation of the CP predicted with the model of Bolshoy et al.

Mentions: The 2D trajectory of Figure 5b was used to simulate the room temperature bending of DNA, describing chain lateral motion onto the mica surface. To this purpose, it was sampled at the spacing (corresponding to the experimentally found helix rise per base-pair) and thermal effects (on bending) were implemented by adding to the angles among neighbor segments a fluctuation chosen by a MC method from normally distributed numbers with mean zero and variance of (). The new trajectories were superimposed on a randomly flat substrate (roughness 0.1 nm) and dilated by a parabolic tip (36) in order to generate topographies resembling as close as possible those obtained by AFM (Figure 6a). These were finally analyzed with the tracing algorithm in order to assure a bias—due to random and systematic angular distortions—comparable to that affecting experimental data. In Figure 6b, we report the obtained results for the two sliding windows of size and , respectively.Figure 6.


Label-free, atomic force microscopy-based mapping of DNA intrinsic curvature for the nanoscale comparative analysis of bent duplexes.

Buzio R, Repetto L, Giacopelli F, Ravazzolo R, Valbusa U - Nucleic Acids Res. (2012)

(a) Representative conformations of six chains generated by MC methods from the intrinsic 2D trajectory predicted by De Santis et al model for the target DNA. A randomly flat substrate has been intentionally added to generate topographies resembling as close as possible those obtained by AFM (b) Theoretical pattern of variation of the CP for two different values of sliding windows length L; encircled are the main peaks of the plots. Experimental results are reported for comparison with L = 34 nm. (c) Intrinsic curvature of the 2D trajectory predicted by De Santis et al model, with marked positions of the pairs of segments of length L = 17 nm (50 bp) related to the peaks highlighted in (b). Positions are shown for clarity also on the 2D chain. (d) Comparison of experimental results with the theoretical pattern of variation of the CP predicted with the model of Bolshoy et al.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367213&req=5

gks210-F6: (a) Representative conformations of six chains generated by MC methods from the intrinsic 2D trajectory predicted by De Santis et al model for the target DNA. A randomly flat substrate has been intentionally added to generate topographies resembling as close as possible those obtained by AFM (b) Theoretical pattern of variation of the CP for two different values of sliding windows length L; encircled are the main peaks of the plots. Experimental results are reported for comparison with L = 34 nm. (c) Intrinsic curvature of the 2D trajectory predicted by De Santis et al model, with marked positions of the pairs of segments of length L = 17 nm (50 bp) related to the peaks highlighted in (b). Positions are shown for clarity also on the 2D chain. (d) Comparison of experimental results with the theoretical pattern of variation of the CP predicted with the model of Bolshoy et al.
Mentions: The 2D trajectory of Figure 5b was used to simulate the room temperature bending of DNA, describing chain lateral motion onto the mica surface. To this purpose, it was sampled at the spacing (corresponding to the experimentally found helix rise per base-pair) and thermal effects (on bending) were implemented by adding to the angles among neighbor segments a fluctuation chosen by a MC method from normally distributed numbers with mean zero and variance of (). The new trajectories were superimposed on a randomly flat substrate (roughness 0.1 nm) and dilated by a parabolic tip (36) in order to generate topographies resembling as close as possible those obtained by AFM (Figure 6a). These were finally analyzed with the tracing algorithm in order to assure a bias—due to random and systematic angular distortions—comparable to that affecting experimental data. In Figure 6b, we report the obtained results for the two sliding windows of size and , respectively.Figure 6.

Bottom Line: We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature.Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far.We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes.

View Article: PubMed Central - PubMed

Affiliation: S.C. Nanobiotecnologie, National Institute for Cancer Research IST, Genova, Italy.

ABSTRACT
We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes.

Show MeSH