title: [Single-molecule detection and characterization of DNA replication based on DNA origami].
abstract: OBJECTIVE
  To investigate single-molecule detection and characterization of DNA replication.
  
  
  METHODS
  Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication.
  
  
  RESULTS
  The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis.
  
  
  CONCLUSIONS
  The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.
authors.affiliation: Physical Biology Laboratory, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China. E-mail: wq2018389@163.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Youjie
authors.initials: Y
authors.lastname: Fan
authors.forename: Bin
authors.initials: B
authors.lastname: Li
mesh_terms: D019921:Biotinylation; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase; D004587:Electrophoresis, Agar Gel; D018625:Microscopy, Atomic Force; D009693:Nucleic Acid Hybridization; D019809:Streptavidin
chemical_list: D004277:DNA, Single-Stranded; D004247:DNA; D019809:Streptavidin; D004259:DNA-Directed DNA Polymerase