Detection Of DNA Replication On DNA Origami By AFM

DNA replication is an essential process of cellular life, and is used in numerous practicalapplications in basic research, clinical diagnosis, and forensics. DNA replication, in other word, isthe synthesis of new double-strand DNA identical with its DNA template. Despite extensive andconsiderable studies, investigation of DNA replication at the single-molecule level remainschallenge. Methods for detecting DNA replication such as how to obtain information about asingle-stranded DNA (ssDNA) turning into a double-stranded DNA (dsDNA) are not available. Inthis study, we directly observe the interaction between DNA and DNA polymerase I fragment(KF)based on the high-resolution capability of atomic force microscopy and the addressable propertyof DNA origami. This paper mainly focuses on the following two aspects:(1) DNA replicationconducted by using a ssDNA with its two ends attached to a DNA origami as the template, anddetection of the resulting of DNA replication;(2)Direct visualization of the movement of a singleKF on a DNA strand in real-time and some factors effect on KF activities.(1) DNA replication conducted by using a ssDNA with its two ends attached to a DNAorigami as the template, and detection of the resulting of DNA replication; after a triangular shapeof DNA origami was constructed, a ssDNA template was attached to the predesigned positions ofDNA origami through hybridization. The data of Gel electrophoresis and AFM indicated that morethan50%ssDNA molecules could be anchored on DNA origami when a mol ratio of the DNAtemplate to DNA origami was more than10:1. It was found that KF could bind to the ssDNAtemplate fixed on DNA origami, and could perform its catalytic activity, leading to the ssDNA intoa dsDNA. Moreover, the resulting dsDNA was subsequently confirmed by a molecular recognitionsystem, the biotin-streptavidin reaction.(2) Direct visualization of the movement of a single KF on a DNA strand in real-time andsome factors effect on KF activities.A series of AFM images directly recorded the catalytic process of KF, including binding, sliding, and dissociation from DNA strand on mica surface. Meanwhile, in the absence of dNTPS,most of KF bound to the locations of5’ end of templates (3’ end of the primers here), whereas inthe existing of dNTPs a gradually decreased binding ratio on the5’ end of the templates suggesteda directional movement of KF. The phenomenon is consisting with the polymeric direction of newDNA strand, from5’ end to3’ end. To test if the method was also suitable for some other lengthsof ssDNA strands. A longer ssDNA template was obtained through changing the sequences ofedge staples and the DNA replication was also achieved. In addition, some factors effected on theactivities of KF was studied.In summary, we detected the DNA replication in a real-time way by combining DNA origamiwith AFM. The method may be valuable for investigation of the mechanism of DNA replicationand interactions between DNA and proteins.