Atomic force microscopy (AFM)-based nanomechanical imaging provides a high-resolution approach for imaging biomolecules with nanometer resolution. Nevertheless, the lack of appropriate nanomechanical labels poses a limit to biological applications. Here, we describe how to generate a set of shape-resolved nanomechanical labels by exploiting self-assembled DNA origami technology. By designing ‘mediator’ strands that can hybridize with both the origami shapes and the target DNA, these origami shape IDs can be used to site-specifically label genomic DNA with high efficiency and high throughput. When DNA origami shape IDs are used to label target sequences containing two single-nucleotide polymorphisms (SNPs), this approach is capable of differentiating adjacent labeling sites separated by only 30 nucleobases (~10 nm) under AFM imaging. This resolution is a threefold improvement of that which can be obtained with imaging-based genotyping using super-resolution imaging. We further demonstrate how to use origami shape IDs for high-resolution genotyping of SNPs in disease-associated genes in patients. The entire protocol takes ~2 d to complete.
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The authors declare no competing interests.
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DNA origami-based shape IDs for single-molecule nanomechanical genotyping: https://doi.org/10.1038/ncomms14738
A DNA-origami chip platform for label-free SNP genotyping using toehold-mediated strand displacement: https://doi.org/10.1002/smll.201000908
Gold nanoparticles for high-throughput genotyping of long-range haplotypes: https://doi.org/10.1038/nnano.2011.141
Asymmetric DNA origami for spatially addressable and index-free solution-phase DNA chips: https://doi.org/10.1002/adma.201000151
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This work was supported by the Ministry of Science and Technology of China (2017YFA0205302, 2016YFA0201200 and 2016YFA0400900), the NSFC (61771253 and 21390414), the Program for Changjiang Scholars and Innovative Research Team in University (IRT 15R37), the Natural Science Foundation of Jiangsu Province (BK20151504) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, YX03001).
Integrated supplementary information
(a) M13mp18 single-strand DNA was annealed with staples in a Peltier thermal cycler (a-1). Then the PCR mixture was purified by agarose gel electrophoresis (a-2) and subsequently extracted using Freeze ’N Squeeze DNA gel extraction spin columns (a-3). (b) phiX 174 DNA was first annealed with mediator primer and extends to dsDNA (b-1). Then the PCR product was purified by agarose gel electrophoresis (b-2) and subsequently extracted using Freeze ’N Squeeze DNA gel extraction spin columns (b-3). The purified target dsDNA hybridized with DNA origami shape IDs (b-4). (c) The process of amplification (c-1), digestion (c-2), annealing and extension (c-3) were used to deal with linear (patient sample) DNA. Then the target DNA was purified by agarose gel electrophoresis (c-4), extracted using Freeze ’N Squeeze DNA gel extraction spin columns (c-5) and hybridized with DNA origami shape IDs (c-6). Finally, all products including DNA origami shape IDs and the mixture products of hybridization were imaged with AFM
The M-strand consists of three blocks, M1, M2 and M3. The M1 block is 20-base long, which is used to hybridize with the target DNA. M1 block is set at the 3′ end of the M-strand and the first base at 3′ end is the complementary base to the target SNP. M2 block is a spacer with five “T”s. M3 block is also 20-base long, which can be recognized by M3’ on the DNA origami shape IDs. Adapted from ref. 6, Springer Nature
(a) Cross-shaped ID with one M3’strand at the corner point. (b) Cross-shaped ID with one M3’strand at the edge middle point. (c) Cross-shaped ID with one M3’strand at the inner point of the origami. Scale bar: 200 nm. Adapted from ref. 6, Springer Nature
(a) Triangular-shaped ID with one M3’strand at the corner point. (b) Triangular-shaped ID with one M3’strand at the edge middle point. (c) Triangular-shaped ID with one M3’strand at the inner point of the origami. Scale bar: 200 nm. Adapted from ref. 6, Springer Nature
(a) Rectangular-shaped ID with one M3’strand at the corner point. (b) Rectangular-shaped ID with one M3’strand at the edge middle point. (c) Rectangular-shaped ID with one M3’strand at the inner point of the origami. Scale bar: 200 nm. Adapted from ref. 6, Springer Nature
Supplementary Fig. 6 Gel electrophoresis of the phiX174 DNA template’s asymmetric extension assisted by AuNPs.
The phiX174 DNA templates were extended by a perfectly matched primer (line T) and one-base mismatched primer (line F) assisted under different concentrations of AuNP. Line M, DL15,000 maker. Adapted from ref. 6, Springer Nature
Supplementary Fig. 7 Gel electrophoresis of regenerated dsDNA from ssDNA for the 4.6-kb AMD samples.
4.6-kb long-range PCR products of four different AMD samples (lanes 1–4) were digested with lambda exonuclease to produce the corresponding ssDNA (lanes 5–8, respectively). After primer extension, dsDNA samples were regenerated (lanes 9–12, respectively). Line M, DL15,000 maker. Adapted from ref. 6, Springer Nature
Supplementary Fig. 8 Gel electrophoresis of regenerated dsDNA from ssDNA for the 12-kb p53 gene samples.
12-kb long-range PCR product of one DNA samples (lanes 1) was digested with lambda exonuclease to produce the corresponding ssDNA (lanes 2). After primer extension, dsDNA samples were regenerated (lanes 3). Line M, DL15,000 maker. Adapted from ref. 6, Springer Nature
Supplementary Fig. 9 Direct haplotyping of 4.6-kb region samples on chromosome 3 and a 12-kb region of the p53 gene from the Han Chinese population by shape IDs.
(a) Upper, schematic showing haplotype and relative locations of two SNPs (rs17038640 (C/T) and rs4676487 (C/T)) on a 4.6-kb region. Lower, for each SNP locus, the triangular- and the STV-decorated triangular-shape IDs are used to label the alleles C and T, respectively. The shape IDs in the AFM images show that the genomic DNA of one sample is homozygous at both loci (C–C), and the genomic DNA of the other three samples is heterozygous at both loci (T–T and C–C). Scale bar, 100 nm. (b) Single-blinded test for haplotyping P53 gene. Upper, schematic showing a 12-kb region of the human p53 gene located on chromosome 17. Each origami shape ID corresponds to a specific allele. For example, for SNP 1, A and C allele correspond to cross-origami with or without STVs, respectively. Middle, AFM images for the haplotypes of these three SNPs. Haplotype 1 contains A–G–T and haplotype 2 contains C–C–C, which are also verified by capillary sequencing data. Lower, when the first SNP was A on one sequence, the other SNPs obtained from independent capillary sequencing were G and T, which was consistent with that from the nanomechanical imaging in haplotype 1. Scale bar, 200 nm. Adapted from ref. 6, Springer Nature
Upper: schematic showing a 34-kb region AMD sample, which is on chromosome 10 from the Han Chinese population. It consists of seven SNP alleles that are split into four overlapping fragments. Each fragment contains two or three SNP loci, which have at least one SNP at the ends as “joint locus”. The “joint locus” is heterozygously shared by its adjacent fragment and used for subsequent fragment connection. Lower: gel electrophoresis of regenerated dsDNA from ssDNA. Four fragments in 34 kb were generated by long-range PCR of one AMD sample (lanes 1, 4, 7 and 10) and were digested with lambda exonuclease to produce the corresponding ssDNA (lanes 2, 5, 8 and 11, respectively). After primer extension, dsDNA samples were regenerated (lanes 3, 6, 9 and 12, respectively). Line M, DL15,000 marker. Adapted from ref. 6, Springer Nature
The long 34-kb region was split into four overlapping fragments. The haplotypes of each fragment can be directly read by shape IDs. For example, in fragment 2, the two haplotypes of C–T–C and T–G–T correspond to SNPs 2, 3 and 4, respectively. Connection of the four fragments reconstructs continuous haplotypes for this 34-kb region. That is, haplotype 1 contains G–C–T–C–A–G–C, and haplotype 2 contains T–T–G–T–G–A–T. All the seven SNPs are heterozygous in the sample, and two haplotypes were read simultaneously using AFM, which were also supported by the capillary sequencing results. Adapted from ref. 6, Springer Nature
Supplementary Figures 1–11, Supplementary Tables 1–3