Limits...
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.

No MeSH data available.


Schematic illustration of AFM-based single-molecule nanomechanical haplotyping with DNA origami shape IDs.(a,b) Diploid genomic DNA extracted from genetic samples, which was site-specifically labeled with ‘multi-colour' shape IDs. The two alleles of each SNP are labeled with different shape IDs. (c) Origami shape IDs serve as ‘magnifying lens' to translate individual SNPs to origami nanostructures of several tens of nanometers. Consequently, the haplotype of this genomic DNA can be directly imaged under AFM. (d) Design and AFM images of sixteen shape IDs using DNA origami decorated with or without STV. Scale bar, 100 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384221&req=5

f1: Schematic illustration of AFM-based single-molecule nanomechanical haplotyping with DNA origami shape IDs.(a,b) Diploid genomic DNA extracted from genetic samples, which was site-specifically labeled with ‘multi-colour' shape IDs. The two alleles of each SNP are labeled with different shape IDs. (c) Origami shape IDs serve as ‘magnifying lens' to translate individual SNPs to origami nanostructures of several tens of nanometers. Consequently, the haplotype of this genomic DNA can be directly imaged under AFM. (d) Design and AFM images of sixteen shape IDs using DNA origami decorated with or without STV. Scale bar, 100 nm.

Mentions: We fabricated a set of shape IDs by using DNA origami designs (Figs 1 and 2, and Supplementary Fig. 1). The basic elements are triangular, cross and rectangular shapes, which are readily distinguishable under AFM imaging. To establish that this shape ID system can specifically target gene sequences, we employed a single-stranded (ss-) bacteriophage phiX 174 DNA with a covalently closed circularity genome of 5,386 nucleotides as the testbed for genetic analysis. The phiX 174 template was first annealed with a three-block ‘mediator' DNA strand (M-strand) that has an M1 block for complementary hybridization with the template, an M2 spacer block and an M3 block for capturing shape IDs (Fig. 2a). Upon hybridization with the template, M1 serves as the primer to initiating DNA extension in the presence of polymerase, which turns the ssDNA template into double-stranded (ds) DNA that is more visible under AFM imaging8. The M3 block is complementary to a short strand M3′ that is carried on each corresponding shape ID.


DNA origami-based shape IDs for single-molecule nanomechanical genotyping
Schematic illustration of AFM-based single-molecule nanomechanical haplotyping with DNA origami shape IDs.(a,b) Diploid genomic DNA extracted from genetic samples, which was site-specifically labeled with ‘multi-colour' shape IDs. The two alleles of each SNP are labeled with different shape IDs. (c) Origami shape IDs serve as ‘magnifying lens' to translate individual SNPs to origami nanostructures of several tens of nanometers. Consequently, the haplotype of this genomic DNA can be directly imaged under AFM. (d) Design and AFM images of sixteen shape IDs using DNA origami decorated with or without STV. Scale bar, 100 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384221&req=5

f1: Schematic illustration of AFM-based single-molecule nanomechanical haplotyping with DNA origami shape IDs.(a,b) Diploid genomic DNA extracted from genetic samples, which was site-specifically labeled with ‘multi-colour' shape IDs. The two alleles of each SNP are labeled with different shape IDs. (c) Origami shape IDs serve as ‘magnifying lens' to translate individual SNPs to origami nanostructures of several tens of nanometers. Consequently, the haplotype of this genomic DNA can be directly imaged under AFM. (d) Design and AFM images of sixteen shape IDs using DNA origami decorated with or without STV. Scale bar, 100 nm.
Mentions: We fabricated a set of shape IDs by using DNA origami designs (Figs 1 and 2, and Supplementary Fig. 1). The basic elements are triangular, cross and rectangular shapes, which are readily distinguishable under AFM imaging. To establish that this shape ID system can specifically target gene sequences, we employed a single-stranded (ss-) bacteriophage phiX 174 DNA with a covalently closed circularity genome of 5,386 nucleotides as the testbed for genetic analysis. The phiX 174 template was first annealed with a three-block ‘mediator' DNA strand (M-strand) that has an M1 block for complementary hybridization with the template, an M2 spacer block and an M3 block for capturing shape IDs (Fig. 2a). Upon hybridization with the template, M1 serves as the primer to initiating DNA extension in the presence of polymerase, which turns the ssDNA template into double-stranded (ds) DNA that is more visible under AFM imaging8. The M3 block is complementary to a short strand M3′ that is carried on each corresponding shape ID.

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.