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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 AFM topography of the target DNA. It shows the persistence of bends at few locations along the molecular backbone—marked by arrows—suggesting the presence of a significant intrinsic curvature at the same places. In the inset is the histogram of contour lengths. (b) Comparison of the experimentally measured end-to-end distance curve with the WLC model predictions for linear (red) and bent (black) chains. The chosen specimen reveals a small but systematic decrease of  at curvilinear distances above 250 nm, ascribed to an overall coiling of the chains with respect to linear DNA of comparable length. The WLC simulations on bent chains are in excellent agreement with experimental data at all curvilinear distances, confirming the key role played by intrinsic curvature.
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gks210-F3: (a) Representative AFM topography of the target DNA. It shows the persistence of bends at few locations along the molecular backbone—marked by arrows—suggesting the presence of a significant intrinsic curvature at the same places. In the inset is the histogram of contour lengths. (b) Comparison of the experimentally measured end-to-end distance curve with the WLC model predictions for linear (red) and bent (black) chains. The chosen specimen reveals a small but systematic decrease of at curvilinear distances above 250 nm, ascribed to an overall coiling of the chains with respect to linear DNA of comparable length. The WLC simulations on bent chains are in excellent agreement with experimental data at all curvilinear distances, confirming the key role played by intrinsic curvature.

Mentions: After samples preparation, a quantitative AFM analysis of molecular profiles was routinely performed in order to test the reproducibility of imaging conditions, evaluate relevant deviations of adsorbed DNA superstructure from the canonical B-form and get deeper insight on the influence of intrinsic curvatures on the local and global geometrical properties of the traced contours. Typically, measured DNA molecules displayed an average width of ∼10 nm and a height of , due respectively to AFM probe convolution effects and to the elastic deformation of the soft molecule under the repulsive forces exerted by the scanning tip (29). Molecules surface density was in the range . The analysis of the contour lengths for a large number of traced molecules (∼400) attested a DNA contraction of 5% with respect to the B-form. This corresponds to a helix rise per base pair of 0.32 nm in excellent agreement with results of similar studies (1,8,10,23,30,31). In Figure 3a we report a representative high resolution topography of the target DNA. As expected, it reveals the large variety of shapes assumed by DNA under the thermal stochastic perturbation of its molecular environment. By visual inspection however, one can already notice the persistence of bends at a few sites, namely in close proximity of both ends and within the central region of the chain. This fact suggests the presence of non- intrinsic curvatures at the same places.Figure 3.


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 AFM topography of the target DNA. It shows the persistence of bends at few locations along the molecular backbone—marked by arrows—suggesting the presence of a significant intrinsic curvature at the same places. In the inset is the histogram of contour lengths. (b) Comparison of the experimentally measured end-to-end distance curve with the WLC model predictions for linear (red) and bent (black) chains. The chosen specimen reveals a small but systematic decrease of  at curvilinear distances above 250 nm, ascribed to an overall coiling of the chains with respect to linear DNA of comparable length. The WLC simulations on bent chains are in excellent agreement with experimental data at all curvilinear distances, confirming the key role played by intrinsic curvature.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks210-F3: (a) Representative AFM topography of the target DNA. It shows the persistence of bends at few locations along the molecular backbone—marked by arrows—suggesting the presence of a significant intrinsic curvature at the same places. In the inset is the histogram of contour lengths. (b) Comparison of the experimentally measured end-to-end distance curve with the WLC model predictions for linear (red) and bent (black) chains. The chosen specimen reveals a small but systematic decrease of at curvilinear distances above 250 nm, ascribed to an overall coiling of the chains with respect to linear DNA of comparable length. The WLC simulations on bent chains are in excellent agreement with experimental data at all curvilinear distances, confirming the key role played by intrinsic curvature.
Mentions: After samples preparation, a quantitative AFM analysis of molecular profiles was routinely performed in order to test the reproducibility of imaging conditions, evaluate relevant deviations of adsorbed DNA superstructure from the canonical B-form and get deeper insight on the influence of intrinsic curvatures on the local and global geometrical properties of the traced contours. Typically, measured DNA molecules displayed an average width of ∼10 nm and a height of , due respectively to AFM probe convolution effects and to the elastic deformation of the soft molecule under the repulsive forces exerted by the scanning tip (29). Molecules surface density was in the range . The analysis of the contour lengths for a large number of traced molecules (∼400) attested a DNA contraction of 5% with respect to the B-form. This corresponds to a helix rise per base pair of 0.32 nm in excellent agreement with results of similar studies (1,8,10,23,30,31). In Figure 3a we report a representative high resolution topography of the target DNA. As expected, it reveals the large variety of shapes assumed by DNA under the thermal stochastic perturbation of its molecular environment. By visual inspection however, one can already notice the persistence of bends at a few sites, namely in close proximity of both ends and within the central region of the chain. This fact suggests the presence of non- intrinsic curvatures at the same places.Figure 3.

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
Related in: MedlinePlus