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DNA origami-based shape IDs for single-molecule nanomechanical genotyping

View Article: PubMed Central - PubMed

ABSTRACT

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

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‘Multi-colour' nanomechanical imaging and genotyping resolution of origami shape IDs.(a) ‘Multi-colour' imaging of phiX174 with multiple shape IDs and their distance analysis. Left, three different shape IDs, triangular (corresponding to site 1,433), triangular with STVs (corresponding to site 1,529) and cross (corresponding to site 4,914), are site-specifically labelled on phiX 174 with different labelling efficiency (85% for the single-site labelling, 56.4% for the two-site labelling and 30.8% for the three-site labelling). Scale bar, 200 nm. Right, histograms for the counts of shape IDs as a function of distance between site 1,433, 1,529 and 4,914 in phiX. As for two-site labelling (left), the measured distance between site 1,433 and 4,914 is 650 nm, which is in good agreement with calculated distance 648 nm. As for three-site labelling (right), the measured distances between site 1,433, 1,529 and 4,914 are 652 nm (blue) and 682 nm (red), which are in good agreement with the calculated distances 648 and 680 nm. Also see Supplementary Figs 14 and 15. (b) Schematic showing and three-dimensional AFM topographic images of triangular- (corresponding to site 1,433), cross- (corresponding to site 1,463) and STV-decorated triangular- (corresponding to site 1,529) shaped IDs for labelling phiX 174. Left, the contour length between two labelled sites (1,433 and 1,463) is ∼10 nm (30 bp). Right, the contour length between two labelled sites (1,433 and 1,529) is ∼32 nm (96 bp).
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f3: ‘Multi-colour' nanomechanical imaging and genotyping resolution of origami shape IDs.(a) ‘Multi-colour' imaging of phiX174 with multiple shape IDs and their distance analysis. Left, three different shape IDs, triangular (corresponding to site 1,433), triangular with STVs (corresponding to site 1,529) and cross (corresponding to site 4,914), are site-specifically labelled on phiX 174 with different labelling efficiency (85% for the single-site labelling, 56.4% for the two-site labelling and 30.8% for the three-site labelling). Scale bar, 200 nm. Right, histograms for the counts of shape IDs as a function of distance between site 1,433, 1,529 and 4,914 in phiX. As for two-site labelling (left), the measured distance between site 1,433 and 4,914 is 650 nm, which is in good agreement with calculated distance 648 nm. As for three-site labelling (right), the measured distances between site 1,433, 1,529 and 4,914 are 652 nm (blue) and 682 nm (red), which are in good agreement with the calculated distances 648 and 680 nm. Also see Supplementary Figs 14 and 15. (b) Schematic showing and three-dimensional AFM topographic images of triangular- (corresponding to site 1,433), cross- (corresponding to site 1,463) and STV-decorated triangular- (corresponding to site 1,529) shaped IDs for labelling phiX 174. Left, the contour length between two labelled sites (1,433 and 1,463) is ∼10 nm (30 bp). Right, the contour length between two labelled sites (1,433 and 1,529) is ∼32 nm (96 bp).

Mentions: Having substantiated the specific gene-targeting ability of individual shape IDs, we next explored differential labelling of a single template using multiple shape IDs. We first expanded the three-element set of shapes by using streptavidin (STV)-decorated origami nanostructures (Supplementary Fig. 13a). By exploiting the addressability of DNA origami, we site-specifically anchored different numbers of STV on prescribed positions carrying biotin tags. STV of ∼4 nm is readily visible as small dots on the origami, which remarkably distinguishes STV-decorated shape IDs from non-decorated ones. Three different shape IDs, triangular, triangular with STV and cross were then chosen to target the sites of 1,433, 1,529 and 4,914 on phiX 174, respectively. We employed multiple ‘mediator' DNA strands to introduce the specific M3 capturing sequences. We found that multiple M1 sequences could initiate simultaneous extension from different sites of phiX 174 in the presence of polymerase, which turned the template into a dsDNA circle similar to the single M1 initiation, albeit with a lower efficiency (Supplementary Figs 13b and 16–20 and Discussion). Importantly, multiple shape IDs can be site-specifically labelled on phiX 174, as illustrated in AFM images (Fig. 3a and Supplementary Figs 18 and 19). We also note that contour lengths between adjacent sites coincide well with those predicated from their base numbers (0.34 nm for one base) for labels at either two or three sites (Supplementary Figs 14 and 15). Of note, whereas the labelling efficiency decreases from single site (∼85%) to two sites (∼56.4%) and three sites (∼30.8%), the single-molecule nature of this nanomechanical analysis allows reliable genotyping even at a low efficiency of 30.8% (Fig. 3a and Supplementary Fig. 20). Analogous to ‘multi-colour' fluorescence imaging-based genetic analysis, differential labelling of multiple shape IDs should greatly improve the genotyping ability in both increasing the accuracy and reducing the labour and time cost. Moreover, accurate distance information between single-nucleotide polymorphism (SNP) sites can be readily obtained from single-molecule-based statistical analysis (Fig. 3a).


DNA origami-based shape IDs for single-molecule nanomechanical genotyping
‘Multi-colour' nanomechanical imaging and genotyping resolution of origami shape IDs.(a) ‘Multi-colour' imaging of phiX174 with multiple shape IDs and their distance analysis. Left, three different shape IDs, triangular (corresponding to site 1,433), triangular with STVs (corresponding to site 1,529) and cross (corresponding to site 4,914), are site-specifically labelled on phiX 174 with different labelling efficiency (85% for the single-site labelling, 56.4% for the two-site labelling and 30.8% for the three-site labelling). Scale bar, 200 nm. Right, histograms for the counts of shape IDs as a function of distance between site 1,433, 1,529 and 4,914 in phiX. As for two-site labelling (left), the measured distance between site 1,433 and 4,914 is 650 nm, which is in good agreement with calculated distance 648 nm. As for three-site labelling (right), the measured distances between site 1,433, 1,529 and 4,914 are 652 nm (blue) and 682 nm (red), which are in good agreement with the calculated distances 648 and 680 nm. Also see Supplementary Figs 14 and 15. (b) Schematic showing and three-dimensional AFM topographic images of triangular- (corresponding to site 1,433), cross- (corresponding to site 1,463) and STV-decorated triangular- (corresponding to site 1,529) shaped IDs for labelling phiX 174. Left, the contour length between two labelled sites (1,433 and 1,463) is ∼10 nm (30 bp). Right, the contour length between two labelled sites (1,433 and 1,529) is ∼32 nm (96 bp).
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f3: ‘Multi-colour' nanomechanical imaging and genotyping resolution of origami shape IDs.(a) ‘Multi-colour' imaging of phiX174 with multiple shape IDs and their distance analysis. Left, three different shape IDs, triangular (corresponding to site 1,433), triangular with STVs (corresponding to site 1,529) and cross (corresponding to site 4,914), are site-specifically labelled on phiX 174 with different labelling efficiency (85% for the single-site labelling, 56.4% for the two-site labelling and 30.8% for the three-site labelling). Scale bar, 200 nm. Right, histograms for the counts of shape IDs as a function of distance between site 1,433, 1,529 and 4,914 in phiX. As for two-site labelling (left), the measured distance between site 1,433 and 4,914 is 650 nm, which is in good agreement with calculated distance 648 nm. As for three-site labelling (right), the measured distances between site 1,433, 1,529 and 4,914 are 652 nm (blue) and 682 nm (red), which are in good agreement with the calculated distances 648 and 680 nm. Also see Supplementary Figs 14 and 15. (b) Schematic showing and three-dimensional AFM topographic images of triangular- (corresponding to site 1,433), cross- (corresponding to site 1,463) and STV-decorated triangular- (corresponding to site 1,529) shaped IDs for labelling phiX 174. Left, the contour length between two labelled sites (1,433 and 1,463) is ∼10 nm (30 bp). Right, the contour length between two labelled sites (1,433 and 1,529) is ∼32 nm (96 bp).
Mentions: Having substantiated the specific gene-targeting ability of individual shape IDs, we next explored differential labelling of a single template using multiple shape IDs. We first expanded the three-element set of shapes by using streptavidin (STV)-decorated origami nanostructures (Supplementary Fig. 13a). By exploiting the addressability of DNA origami, we site-specifically anchored different numbers of STV on prescribed positions carrying biotin tags. STV of ∼4 nm is readily visible as small dots on the origami, which remarkably distinguishes STV-decorated shape IDs from non-decorated ones. Three different shape IDs, triangular, triangular with STV and cross were then chosen to target the sites of 1,433, 1,529 and 4,914 on phiX 174, respectively. We employed multiple ‘mediator' DNA strands to introduce the specific M3 capturing sequences. We found that multiple M1 sequences could initiate simultaneous extension from different sites of phiX 174 in the presence of polymerase, which turned the template into a dsDNA circle similar to the single M1 initiation, albeit with a lower efficiency (Supplementary Figs 13b and 16–20 and Discussion). Importantly, multiple shape IDs can be site-specifically labelled on phiX 174, as illustrated in AFM images (Fig. 3a and Supplementary Figs 18 and 19). We also note that contour lengths between adjacent sites coincide well with those predicated from their base numbers (0.34 nm for one base) for labels at either two or three sites (Supplementary Figs 14 and 15). Of note, whereas the labelling efficiency decreases from single site (∼85%) to two sites (∼56.4%) and three sites (∼30.8%), the single-molecule nature of this nanomechanical analysis allows reliable genotyping even at a low efficiency of 30.8% (Fig. 3a and Supplementary Fig. 20). Analogous to ‘multi-colour' fluorescence imaging-based genetic analysis, differential labelling of multiple shape IDs should greatly improve the genotyping ability in both increasing the accuracy and reducing the labour and time cost. Moreover, accurate distance information between single-nucleotide polymorphism (SNP) sites can be readily obtained from single-molecule-based statistical analysis (Fig. 3a).

View Article: PubMed Central - PubMed

ABSTRACT

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

No MeSH data available.


Related in: MedlinePlus