Research And Application Of Precise Genetic Detection Based On DNA Nanotechnology And High-resolution Atomic Force Microscopy Imaging

DNA nanotechnology is a new discipline in the field of nanoscience.Based on the principle of Watson-Click base pairing rules,DNA can be constructed to spatial structure of various dimensions through self-assembly,which holding great promise for the application on different fields in the future.Since the ‘Holiday-junction' structure constructed by N.C.Seeman in 1983,who is the pineenor in the field of nanoscience,DNA nanotechnology has developing for 35 years.It includes DNA tile self-assembly,DNA origami,DNA nanodevice application and so on.Comparing with traditional nanomaterials,DNA nanomaterials is more and more popuplar in the application of mathematics,physics,chemistry,computer science and biomedicine due to its incomparable superiority.By reviewing the development history of DNA nanotechnology,this paper discussed the current advances of DNA nanotechnology in various fields and focuses on the application of biomedicine,especially in genetics.For the further understanding and development of current DNA self-assembly technology,we devoted to solve the key problems in biomedical and precision medicine.The research was carried out mainly includes the following aspects:PART I-Single molecule haplotyping based on DNA origami probes.With the completion of the International Human Genome Project and the rapid development of the high-throughput sequencing technologies,developing effective genetic variation detection and analysis methods is becoming more and more necessary for translational medicine and precision medicine.However,revealing long range haplotype information,which holds great promise for identifying genetic causal variants of complex disorders,is still a challage due to the restriction of sequcing short-reads.Traditional approaches,such as simulation calculation or Allele-specific polymerase chain reaction(AS-PCR),made it difficult to obtain genomic haplotype information either for the inaccurate simulation or the complexity of experimental techniques.Here we developed 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 more than 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.PART II-Identifying the genotypes of hepatitis B virus(HBV)with DNA origami label.Based on the previous work of haplotyping target genes with DNA origami probes,we next explored the application of DNA nanotechnology in precision medicine.Precise diagnose,prediction of diseases and improving the survival of patients are the major challenges in biomedical research today.Improving the specificity and sensitivity of genetic detection approach is helpful for this problem.The traditional methods(such as PCR,RT-PCR or sequencing)can only reveal the superposed signal by the amplification of DNA molecules,while the partial mutation occurred during the disease outbreaks may be ignored.The hepatitis B virus(HBV)genotyping may profoundly affect the accurate diagnosis and antiviral treatment of viral hepatitis.Existing genotyping methods such as serological,immunological,or molecular testing are still suffered substandard specificity and low sensitivity in laboratory or clinical application.Therefore,as a further confirmatory research to explore the sensitivity and applicability of this assay,differentially pre-designed DNA origami shape IDs probes were developed for precisely HBV genotyping.Through the specific identification of visualized DNA origami nanostructure with clinical HBV DNA samples,the genetic variations information of genotypes can be directly identified under AFM.As a proof-of-concept,five genotype B and six genotype C were detected in eleven HBV infected patients' blood DNA samples of Han Chinese population in the single-blinded test with high-specificity.Meanwhile,sensitivity test were performed with a detection limit down to 10 pM that superior than most of the traditional assays.Our findings confirmed that this ultrahigh-resolution origami shape IDs imaging-based genetic analytic strategy is a robust,specific and effective approach as described in our previous research,moreover,with great sensitivity on the limit of detection for the quantitative detection of virus infection.With the rapid development and application of AFM in both resolution and throughput,it is possible that the detection limit may be much lower and even only single DNA molecule copy in the future,holding great promise of early virus-infection diagnosis and targeted medical therapeutics for clinical translation.In the last part,we make some prospects for the future development of DNA nanotechnology and introduce several directions that may be applied in the future.Although the current research progress of DNA nanotechnology is not as mature as traditional nanomaterials,it is true that DNA nanomaterials has became more and more popular and holds great promise in various scientific research fields.