The Discovery of Rolling Circle Amplification and Rolling Circle Transcription

Abstract

CONSPECTUS Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25–100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase “rolling circle” activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential (“hyperbranched”) RCA amplification made possible by use of a second primer; the development of the “padlock” process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.

Re-evaluating the conventional wisdom about binding assays

“…The authors used a three-state population-shift mechanism, which they termed recognition-enhanced metastably-shielded aptamer probes (RMSApt). The affinity reagents are designed in such a way that they can initiate a rolling circle amplification (RCA) reaction [35], [36] when bound to the target molecule. They are coupled to a solid surface, so that individual RCA products can be imaged and counted.…”

Rolling Circle Amplification is a high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

“…In this report, we describe an alternative to bacterial-based growth of infectious cDNA clones-in vitro amplification using rolling circle amplification (RCA). RCA is an isothermal, high yield method of DNA amplification [19] that uses a highly processive polymerase that can amplify DNA over 70kb [20]. Importantly, the enzyme replicates DNA with high fidelity due to its 3'-5'exonuclease-or proofreading-activity [21].…”

“…These data demonstrate that RCA is equivalent to plasmids in their ability to rescue virus using standard transfection conditions in terms of viral yield. A hypothesis for the delay in viral production seen in BHK and HEK293T cells is that the complex structure of RCA products (i.e., branched RCA molecules) compared to plasmid DNA may produce steric inhibition and delay transcription from the CMV promoter by RNA polymerase II [19].…”

Nucleic Acid–Based Functional Nanomaterials as Advanced Cancer Therapeutics

“…Similar to RCA, RCT produced long single‐strand RNA with preprogrammed circular DNA template in the presence of RNA polymerases through rolling circle amplification . As an efficient enzymatic reaction to generate multiple as well as repetitive designed RNA sequences, RCT has also applied to the field of siRNA delivery …”