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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) Main steps of curvature analysis for a DNA chain. A molecule is imaged by AFM then traced by an image-analysis software and represented as a chain of xy pairs separated by a contour length l. The signed bending angle  is obtained from the vector product of the local tangent vectors  and . (b) Generally speaking, we can ascribe four different spatial orientations to the extracted contour of a label-free molecule, according to the end chosen as the starting point of the nucleotide sequence (red dot) and the molecular face exposed to the substrate. As a result, the signed curvature  changes in modulus and/or sign according to the chosen orientation. On the contrary, the CP  is estimated by coupling the signed curvatures of two m-units long segments, symmetrically placed at j units from chain ends. Such quantity remains the same for each one of the possible orientations of the extracted contour. (c) The characteristic patterns of variation of  for some specimens—here named (1–3) —enable to highlight DNA regions with different intrinsic curvature (gray box). This represents an original strategy to establish the comparative analysis of bent duplexes under label-free conditions.
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gks210-F2: (a) Main steps of curvature analysis for a DNA chain. A molecule is imaged by AFM then traced by an image-analysis software and represented as a chain of xy pairs separated by a contour length l. The signed bending angle is obtained from the vector product of the local tangent vectors and . (b) Generally speaking, we can ascribe four different spatial orientations to the extracted contour of a label-free molecule, according to the end chosen as the starting point of the nucleotide sequence (red dot) and the molecular face exposed to the substrate. As a result, the signed curvature changes in modulus and/or sign according to the chosen orientation. On the contrary, the CP is estimated by coupling the signed curvatures of two m-units long segments, symmetrically placed at j units from chain ends. Such quantity remains the same for each one of the possible orientations of the extracted contour. (c) The characteristic patterns of variation of for some specimens—here named (1–3) —enable to highlight DNA regions with different intrinsic curvature (gray box). This represents an original strategy to establish the comparative analysis of bent duplexes under label-free conditions.

Mentions: Common practice in AFM studies on DNA structure and flexibility dictates to prepare specimens by DNA adsorption from an aqueous solution onto an atomically flat substrate. This is followed by high-resolution imaging of adsorbed species and by the use of an image-analysis software in order to reconstruct the molecular profiles and analyze the signed curvature associated to segments of given location and length (2–5,8,24). Tracing algorithms represent each molecule as a chain of xy pairs separated by a fixed contour length l. The curvature analysis along a generic trajectory proceeds through the calculation of the signed bending angles formed by the adjacent units, that are obtained from the vector product of the local tangent vectors and ( with N total number of units) (3). From the values one can define the global curvature for a segment of m units, located at units from one of the ends, as:(1)with (Figure 2a).Figure 2.


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) Main steps of curvature analysis for a DNA chain. A molecule is imaged by AFM then traced by an image-analysis software and represented as a chain of xy pairs separated by a contour length l. The signed bending angle  is obtained from the vector product of the local tangent vectors  and . (b) Generally speaking, we can ascribe four different spatial orientations to the extracted contour of a label-free molecule, according to the end chosen as the starting point of the nucleotide sequence (red dot) and the molecular face exposed to the substrate. As a result, the signed curvature  changes in modulus and/or sign according to the chosen orientation. On the contrary, the CP  is estimated by coupling the signed curvatures of two m-units long segments, symmetrically placed at j units from chain ends. Such quantity remains the same for each one of the possible orientations of the extracted contour. (c) The characteristic patterns of variation of  for some specimens—here named (1–3) —enable to highlight DNA regions with different intrinsic curvature (gray box). This represents an original strategy to establish the comparative analysis of bent duplexes under label-free conditions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks210-F2: (a) Main steps of curvature analysis for a DNA chain. A molecule is imaged by AFM then traced by an image-analysis software and represented as a chain of xy pairs separated by a contour length l. The signed bending angle is obtained from the vector product of the local tangent vectors and . (b) Generally speaking, we can ascribe four different spatial orientations to the extracted contour of a label-free molecule, according to the end chosen as the starting point of the nucleotide sequence (red dot) and the molecular face exposed to the substrate. As a result, the signed curvature changes in modulus and/or sign according to the chosen orientation. On the contrary, the CP is estimated by coupling the signed curvatures of two m-units long segments, symmetrically placed at j units from chain ends. Such quantity remains the same for each one of the possible orientations of the extracted contour. (c) The characteristic patterns of variation of for some specimens—here named (1–3) —enable to highlight DNA regions with different intrinsic curvature (gray box). This represents an original strategy to establish the comparative analysis of bent duplexes under label-free conditions.
Mentions: Common practice in AFM studies on DNA structure and flexibility dictates to prepare specimens by DNA adsorption from an aqueous solution onto an atomically flat substrate. This is followed by high-resolution imaging of adsorbed species and by the use of an image-analysis software in order to reconstruct the molecular profiles and analyze the signed curvature associated to segments of given location and length (2–5,8,24). Tracing algorithms represent each molecule as a chain of xy pairs separated by a fixed contour length l. The curvature analysis along a generic trajectory proceeds through the calculation of the signed bending angles formed by the adjacent units, that are obtained from the vector product of the local tangent vectors and ( with N total number of units) (3). From the values one can define the global curvature for a segment of m units, located at units from one of the ends, as:(1)with (Figure 2a).Figure 2.

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