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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) Representation of the average 3D shape of two homozygous samples according to the wedge model by De Santis et al. The SNP at the sequence site 762 (marked by arrows) impacts the overall relative orientation of the 5′ half with respect to the 3′ end, whereas the other three SNPs do not substantially affect the DNA shape. (b) Top: theoretical patterns of variation of the CP for the two chains in (a) with L = 34 nm. Bottom: experimental patterns of variation for the CP. Gray regions highlight statistically relevant differences between the two specimens, in excellent agreement with theoretical predictions.
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gks210-F8: (a) Representation of the average 3D shape of two homozygous samples according to the wedge model by De Santis et al. The SNP at the sequence site 762 (marked by arrows) impacts the overall relative orientation of the 5′ half with respect to the 3′ end, whereas the other three SNPs do not substantially affect the DNA shape. (b) Top: theoretical patterns of variation of the CP for the two chains in (a) with L = 34 nm. Bottom: experimental patterns of variation for the CP. Gray regions highlight statistically relevant differences between the two specimens, in excellent agreement with theoretical predictions.

Mentions: One interesting possibility of application might regard the systematic use of CP maps to deeply explore the predictions of DNA adsorption and bending models. An insight into this topic was provided in the sections above and significant improvements are expected to come from state of the art modeling (as Brownian dynamics and molecular dynamics simulations) going beyond the nearest-neighbor approximation in conformational analysis or describing the non-equilibrium processes of DNA adsorption and relaxation on the atomically flat substrate (24,35,37,38). For example, a tight comparison of experimental and theoretical CP patterns might allow to identify the presence of restricted regions where out-of-equilibrium alterations of the chain architecture systematically take place during adsorption. This information might be eventually related to the local base pairs sequence and/or exploited to tune DNA adsorption according to the needs of novel comparative essays. Another challenge might involve the use of CP patterns to routinely detect small conformational changes in large sample numbers. The capability to relate DNA structural variations to physical or biological causes (e.g. mutations at one or more base-pairs) might eventually contribute to develop new assays and even genetic screening protocols for highly bent duplexes. Interestingly, some studies might explore the ultimate sensitivity of CP patterns to point mutations and mismatched base-pairs and largely contribute to the discovery of physical methodologies for molecular haplotyping (16,39). Within this context we offer a concrete example on the CP patterns sensitivity to single nucleotide polymorphisms (SNPs) in the OPN encoding gene. In detail, we contrast two homozygous specimens having different SNPs at four, well-known polymorphic sites. To date, there is a well documented functional effect of such SNPs on the OPN gene transcriptional activity (22), and they play a useful role as genetic markers to characterize patients with oligoarticular juvenile idiopathic arthritis (40). In Figure 8a, we show the 3D model chains predicted for the two specimens. It appears that the insertion (or deletion) of an individual G base at the sequence site 762 (marked by the vertical arrows) dramatically affects the whole DNA bending close to the centre of the chain, in fact inducing a variation of the relative orientation of the 5′ half with respect to the 3′ end. This is confirmed by the corresponding CP patterns, evaluated through theory and experiment as described above. In particular, Figure 8b attests the emergence of statistically relevant differences for the experimental CP values at four main regions of the curvilinear distance s (highlighted in gray). The experimental pattern of the 1335 bp specimen also shows less marked amplitude variations with respect to the 1332 bp counterpart. A detailed analysis of the overall fluctuation of the CP signal for different L values (50–120 bp) confirms that this feature systematically occurs in both theory and experiment (see Supplementary Data); it thus represents a robust sequence-dependent property of the samples that is successfully captured by the CP method. We underline that the most relevant message of Figure 8b is to document the practical feasibility of the label-free comparative analysis envisaged in Figure 2c. Such an essay represents the crucial advantage offered by the CP method with respect to other conformational methods and is reported in the present investigation—to the best of our knowledge—for the first time.Figure 8.


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) Representation of the average 3D shape of two homozygous samples according to the wedge model by De Santis et al. The SNP at the sequence site 762 (marked by arrows) impacts the overall relative orientation of the 5′ half with respect to the 3′ end, whereas the other three SNPs do not substantially affect the DNA shape. (b) Top: theoretical patterns of variation of the CP for the two chains in (a) with L = 34 nm. Bottom: experimental patterns of variation for the CP. Gray regions highlight statistically relevant differences between the two specimens, in excellent agreement with theoretical predictions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks210-F8: (a) Representation of the average 3D shape of two homozygous samples according to the wedge model by De Santis et al. The SNP at the sequence site 762 (marked by arrows) impacts the overall relative orientation of the 5′ half with respect to the 3′ end, whereas the other three SNPs do not substantially affect the DNA shape. (b) Top: theoretical patterns of variation of the CP for the two chains in (a) with L = 34 nm. Bottom: experimental patterns of variation for the CP. Gray regions highlight statistically relevant differences between the two specimens, in excellent agreement with theoretical predictions.
Mentions: One interesting possibility of application might regard the systematic use of CP maps to deeply explore the predictions of DNA adsorption and bending models. An insight into this topic was provided in the sections above and significant improvements are expected to come from state of the art modeling (as Brownian dynamics and molecular dynamics simulations) going beyond the nearest-neighbor approximation in conformational analysis or describing the non-equilibrium processes of DNA adsorption and relaxation on the atomically flat substrate (24,35,37,38). For example, a tight comparison of experimental and theoretical CP patterns might allow to identify the presence of restricted regions where out-of-equilibrium alterations of the chain architecture systematically take place during adsorption. This information might be eventually related to the local base pairs sequence and/or exploited to tune DNA adsorption according to the needs of novel comparative essays. Another challenge might involve the use of CP patterns to routinely detect small conformational changes in large sample numbers. The capability to relate DNA structural variations to physical or biological causes (e.g. mutations at one or more base-pairs) might eventually contribute to develop new assays and even genetic screening protocols for highly bent duplexes. Interestingly, some studies might explore the ultimate sensitivity of CP patterns to point mutations and mismatched base-pairs and largely contribute to the discovery of physical methodologies for molecular haplotyping (16,39). Within this context we offer a concrete example on the CP patterns sensitivity to single nucleotide polymorphisms (SNPs) in the OPN encoding gene. In detail, we contrast two homozygous specimens having different SNPs at four, well-known polymorphic sites. To date, there is a well documented functional effect of such SNPs on the OPN gene transcriptional activity (22), and they play a useful role as genetic markers to characterize patients with oligoarticular juvenile idiopathic arthritis (40). In Figure 8a, we show the 3D model chains predicted for the two specimens. It appears that the insertion (or deletion) of an individual G base at the sequence site 762 (marked by the vertical arrows) dramatically affects the whole DNA bending close to the centre of the chain, in fact inducing a variation of the relative orientation of the 5′ half with respect to the 3′ end. This is confirmed by the corresponding CP patterns, evaluated through theory and experiment as described above. In particular, Figure 8b attests the emergence of statistically relevant differences for the experimental CP values at four main regions of the curvilinear distance s (highlighted in gray). The experimental pattern of the 1335 bp specimen also shows less marked amplitude variations with respect to the 1332 bp counterpart. A detailed analysis of the overall fluctuation of the CP signal for different L values (50–120 bp) confirms that this feature systematically occurs in both theory and experiment (see Supplementary Data); it thus represents a robust sequence-dependent property of the samples that is successfully captured by the CP method. We underline that the most relevant message of Figure 8b is to document the practical feasibility of the label-free comparative analysis envisaged in Figure 2c. Such an essay represents the crucial advantage offered by the CP method with respect to other conformational methods and is reported in the present investigation—to the best of our knowledge—for the first time.Figure 8.

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