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. Conclusion 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 DNApolymerase and the factors affecting DNAreplication.