Rolling circle replication

Abstract: We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and phi29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.

Abstract: It is now clear that there is a diversity of circular RNAs in biological systems. Circular RNAs can be produced by the direct ligation of 5′ and 3′ ends of linear RNAs, as intermediates in RNA processing reactions, or by “backsplicing,” wherein a downstream 5′ splice site (splice donor) is joined to an upstream 3′ splice site (splice acceptor). Circular RNAs have unique properties including the potential for rolling circle amplification of RNA, the ability to rearrange the order of genomic information, protection from exonucleases, and constraints on RNA folding. Circular RNAs can function as templates for viroid and viral replication, as intermediates in RNA processing reactions, as regulators of transcription in cis, as snoRNAs, and as miRNA sponges. Herein, we review the breadth of circular RNAs, their biogenesis and metabolism, and their known and anticipated functions.

Abstract: Rolling circle amplification (RCA) is an isothermal enzymatic process where a short DNA or RNA primer is amplified to form a long single stranded DNA or RNA using a circular DNA template and special DNA or RNA polymerases. The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. The power, simplicity, and versatility of the DNA amplification technique have made it an attractive tool for biomedical research and nanobiotechnology. Traditionally, RCA has been used to develop sensitive diagnostic methods for a variety of targets including nucleic acids (DNA, RNA), small molecules, proteins, and cells. RCA has also attracted significant attention in the field of nanotechnology and nanobiotechnology. The RCA-produced long, single-stranded DNA with repeating units has been used as template for the periodic assembly of nanospecies. Moreover, since RCA products can be tailor-designed by manipulating the circular template, RCA has been employed to generate complex DNA nanostructures such as DNA origami, nanotubes, nanoribbons and DNA based metamaterials. These functional RCA based nanotechnologies have been utilized for biodetection, drug delivery, designing bioelectronic circuits and bioseparation. In this review, we introduce the fundamental engineering principles used to design RCA nanotechnologies, discuss recently developed RCA-based diagnostics and bioanalytical tools, and summarize the use of RCA to construct multivalent molecular scaffolds and nanostructures for applications in biology, diagnostics and therapeutics.

Abstract: Natural genes and proteins often contain tandemly repeated sequence motifs that dramatically increase physiological specificity and activity. Given the selective value of such repeats, it is likely that several different mechanisms have been responsible for their generation. One mechanism that has been shown to generate relatively long tandem repeats (in the kilobase range) is rolling circle replication. In this communication, we demonstrate that rolling circle synthesis in a simple enzymatic system can produce tandem repeats of monomers as short as 34 bp. In addition to suggesting possible origins for natural tandem repeats, these observations provide a facile means for constructing libraries of repeated motifs for use in "in vitro evolution" experiments designed to select molecules with defined biological or chemical properties.