Репликация круговой ДНК: механизмы и применение

DNA synthesis technique

Rolling circle replication (RCR) is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses also replicate their DNA or RNA via the rolling circle mechanism. As a simplified version of natural rolling circle replication, an isothermal DNA amplification technique, rolling circle amplification was developed. The RCA mechanism is widely used in molecular biology and biomedical nanotechnology, especially in the field of biosensing (as a method of signal amplification).

Some DNA viruses replicate their genomic information in host cells via rolling circle replication. For instance, human herpesvirus 6 (HHV 6)(hibv) expresses a set of "early genes" that are believed to be involved in this process. The long concatemers that result are subsequently cleaved between the pac 1 and pac 2 regions of HHV 6's genome by ribozymes when it is packaged into individual virions. Human Papillomavirus 16 (HPV 16) is another virus that employs rolling replication to produce progeny at a high rate. HPV 16 infects human epithelial cells and has a double stranded circular genome. During replication, at the origin, the E1 hexamer wraps around the single strand DNA and moves in the 3' to 5' direction. In normal bidirectional replication, the two replication proteins will disassociate at time of collision, but in HPV 16 it is believed that the E1 hexamer does not disassociate, hence leading to a continuous rolling replication. It is believed that this replication mechanism of HPV may have physiological implications into the integration of the virus into the host chromosome and eventual progression into cervical cancer. In addition, geminivirus also utilizes rolling circle replication as its replication mechanism. It is a virus that is responsible for destroying many major crops, such as cassava, cotton, legumes, maize, tomato and okra. The virus has a circular, single stranded, DNA that replicates in host plant cells. The entire process is initiated by the geminiviral replication initiator protein, Rep, which is also responsible for altering the host environment to act as part of the replication machinery. Rep is also strikingly similar to most other rolling replication initiator proteins of eubacteria, with the presence of motifs I, II, and III at is N terminus. During the rolling circle replication, the ssDNA of geminivirus is converted to dsDNA and Rep is then attached to the dsDNA at the origin sequence TAATATTAC. After Rep, along with other replication proteins, binds to the dsDNA it forms a stem loop where the DNA is then cleaved at the nanomer sequence causing a displacement of the strand. This displacement allows the replication fork to progress in the 3’ to 5’ direction which ultimately yields a new ssDNA strand and a concatameric DNA strand. Bacteriophage T4 DNA replication intermediates include circular and branched circular concatemeric structures. These structures likely reflect a rolling circle mechanism of replication.

Some RNA viruses and viroids also replicate their genome through rolling circle RNA replication. For viroids, there are two alternative RNA replication pathways that respectively followed by members of the family Pospivirodae (asymmetric replication) and Avsunviroidae (symmetric replication). In the family Pospiviroidae (PSTVd like), the circular plus strand RNA is transcribed by a host RNA polymerase into oligomeric minus strands and then oligomeric plus strands. These oligomeric plus strands are cleaved by a host RNase and ligated by a host RNA ligase to reform the monomeric plus strand circular RNA. This is called the asymmetric pathway of rolling circle replication. The viroids in the family Avsunviroidae (ASBVd like) replicate their genome through the symmetric pathway of rolling circle replication. In this symmetric pathway, oligomeric minus strands are first cleaved and ligated to form monomeric minus strands, and then are transcribed into oligomeric plus strands. These oligomeric plus strands are then cleaved and ligated to reform the monomeric plus strand. The symmetric replication pathway was named because both plus and minus strands are produced the same way. Cleavage of the oligomeric plus and minus strands is mediated by the self cleaving hammerhead ribozyme structure present in the Avsunviroidae, but such structure is absent in the Pospiviroidae.

RCA can amplify a single molecular binding event over a thousandfold, making it particularly useful for detecting targets with ultra low abundance. RCA reactions can be performed in not only free solution environments, but also on a solid surface like glass, micro or nano bead, microwell plates, microfluidic devices or even paper strips. This feature makes it a very powerful tool for amplifying signals in solid phase immunoassays (e. g., ELISA). In this way, RCA is becoming a highly versatile signal amplification tool with wide ranging applications in genomics, proteomics, diagnosis and biosensing.

In addition to antibody mediated immuno RCA, the ssDNA RCA primer can be conjugated to the 3' end of a DNA aptamer as well. The primer tail can be amplified through rolling circle amplification. The product can be visualized through the labeling of fluorescent reporter. The process is illustrated in the figure on the right.

Various derivatives of RCA were widely used in the field of biosensing. For example, RCA has been successfully used for detecting the existence of viral and bacterial DNA from clinical samples, which is very beneficial for rapid diagnostics of infectious diseases. It has also been used as an on chip signal amplification method for nucleic acid (for both DNA and RNA) microarray assay. and formation of nano islands.