Rolling circle amplification of synthetic DNA accelerates biocatalytic determination of enzyme activity relative to conventional methods.

The ability to quickly and easily assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Cell-free synthesis, although not practically amenable for large-scale enzyme production, provides a way to accelerate the timeline for screening enzyme candidates using small-scale reactions. However, because cell-free enzyme synthesis requires a considerable amount of template DNA, the preparation of high-quality DNA "parts" in large quantities represents a costly and rate-limiting prerequisite for high throughput screening. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time. We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. This method provides a new alternative to more cumbersome plasmid- or PCR-based protein expression workflows and should be amenable to automation for accelerating enzyme screening in industrial applications.

Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle.

Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed molecules, even from a single DNA molecule, with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular molecules constructed by a multi-fragment assembly reaction. Our results define the minimum element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA molecules without relying on conventional biological cloning.

[Synthesis of Circular DNA Templates with T4 RNA Ligase for Rolling Circle Amplification].

Currently, isothermal methods of nucleic acid amplification have been well established; in particular, rolling circle amplification is of great interest. In this approach, circular ssDNA molecules have been used as a target that can be obtained by the intramolecular template-dependent ligation of an oligonucleotide C-probe. Here, a new method of synthesizing small circular DNA molecules via the cyclization of ssDNA based on T4 RNA ligase has been proposed. Circular ssDNA is further used as the template for the rolling circle amplification. The maximum yield of the cyclization products was observed in the presence of 5-10% polyethylene glycol 4000, and the optimum DNA length for the cyclization constituted 50 nucleotides. This highly sensitive method was shown to detect less than 10^(2) circular DNA molecules. The method reliability was proved based on artificially destroyed dsDNA, which suggests its implementation for analyzing any significantly fragmented dsDNA.

The Discovery of Rolling Circle Amplification and Rolling Circle Transcription.

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.

Circular DNA by "Bis-Click" Ligation: Template-Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides.

A highly effective and convenient "bis-click" strategy was developed for the template-independent circularization of single-stranded oligonucleotides by employing copper(I)-assisted azide-alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7-deaza-2'-deoxyadenosine and 2'-deoxyuridine residues with different side chains were used in solid-phase synthesis with phosphoramidite chemistry. The bis-click ligation of linear 9- to 36-mer oligonucleotides with 1,4-bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9-mers. Compared with linear duplexes, circular bis-click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis-click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides.

Helix untwisting and bubble formation in circular DNA.

The base pair fluctuations and helix untwisting are examined for a circular molecule. A realistic mesoscopic model including twisting degrees of freedom and bending of the molecular axis is proposed. The computational method, based on path integral techniques, simulates a distribution of topoisomers with various twist numbers and finds the energetically most favorable molecular conformation as a function of temperature. The method can predict helical repeat, openings loci, and bubble sizes for specific sequences in a broad temperature range. Some results are presented for a short DNA circle recently identified in mammalian cells.

Condensation of circular DNA.

A simple model of a circularly closed double-stranded DNA in a poor solvent is considered as an example of a semi-flexible polymer with self-attraction. To find the ground states, the conformational energy is computed as a sum of the bending and torsional elastic components and the effective self-attraction energy. The model includes a relative orientation or sequence dependence of the effective attraction forces between different pieces of the polymer chain. Two series of conformations are analysed: a multicovered circle (a toroid) and a multifold two-headed racquet. The results are presented as a diagram of state. It is suggested that the stability of particular conformations may be controlled by proper adjustment of the primary structure. Application of the model to other semi-flexible polymers is considered.

Circular single-stranded synthetic DNA delivery vectors for microRNA.

Single-stranded (ss) circular oligodeoxynucleotides were previously found to undergo rolling circle transcription (RCT) by phage and bacterial RNA polymerases (RNAPs) into tandemly repetitive RNA multimers. Here, we redesign them to encode minimal primary miRNA mimics, with the long term aim of intracellular transcription followed by RNA processing and maturation via endogenous pathways. We describe an improved method for circularizing ss synthetic DNA for RCT by using a recently described thermostable RNA ligase, which does not require a splint oligonucleotide to juxtapose the ligating ends. In vitro transcription of four templates demonstrates that the secondary structure inherent in miRNA-encoding vectors does not impair their RCT by RNAPs previously shown to carry out RCT. A typical primary-miRNA rolling circle transcript was accurately processed by a human Drosha immunoprecipitate, indicating that if human RNAPs prove to be capable of RCT, the resulting transcripts should enter the endogenous miRNA processing pathway in human cells. Circular oligonucleotides are therefore candidate vectors for small RNA delivery in human cells, which express RNAPs related to those tested here.

Covalently linked DNA nanotubes.

The present study introduces an approach to prepare covalently linked DNA nanotubes. A circular DNA that includes at its opposite poles thiol and amine functionalities acts as the building block for the construction of the DNA nanotubes. The circular DNA is cross-linked with a bis-amide-modified nucleic acid to yield DNA nanowires, and these are subsequently cross-linked by a bis-thiolated nucleic acid to yield the DNA nanotubes. Alternatively, a circular DNA that includes four amine functionalities on its poles is cross-linked in one-step by the bis-thiolated nucleic acid to yield the nanotubes. The resulting nanostructures are stable and nonseparable upon heating.

A novel approach to the synthesis of DNA and RNA lariats.

Current studies of lariat RNA structure and function are hindered by the lack of access to synthetic lariats. A novel approach to the synthesis of both DNA and RNA lariats is presented here. Noteworthy features of the methodology are the regiospecific formation of the 2'-5'-phosphodiester linkage, the unusual parallel stranded DNA/RNA hybrid (or parallel RNA/RNA duplex) that forms between an RNA template and a folded 22-nt DNA (or RNA) substrate, and the efficiency of the chemical ligation step at an adenosine branchpoint (50-80%). The DNA and RNA lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition were confirmed by MALDI-TOF mass spectrometry. Thermal denaturation as well as enzymatic and chemical hydrolysis fully supported the proposed lariat structures. Characterization of control parallel duplexes was conducted by gel shift assays and enzymatic degradation with RNase H. The successful synthesis of the lariat molecules described here will allow structural and biochemical studies aimed at better understanding the splicing and debranching mechanisms in which these unusual nucleic acids are involved.

Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry.

The copper-catalyzed azide-alkyne cycloaddition reaction has been used for the template-mediated chemical ligation of two oligonucleotide strands, one with a 5'-alkyne and the other with a 3'-azide, to produce a DNA strand with an unnatural backbone at the ligation point. A template-free click-ligation reaction has been used for the intramolecular circularization of a single stranded oligonucleotide which was used as a template for the synthesis of a covalently closed DNA catenane.

Stability of chitosan-pDNA complex powder prepared by supercritical carbon dioxide process.

The present study examined the stability of a gene in powders prepared with supercritical carbon dioxide (CO(2)) from the viewpoints of the ternary structure of DNA and in vivo transfection potential. An aqueous chitosan-pCMV-Luc complex solution containing mannitol was injected into the stream of a supercritical CO(2)/ethanol admixture to precipitate a gene powder. The obtained gene powders and gene solutions were placed in stability chambers at 25 or 40 degrees C for 4 weeks. The integrity and transfection potency of the gene were examined by electrophoresis and in vivo pulmonary transfection study in mice. The supercritical CO(2) process decreased the supercoiled DNA during the manufacturing process; however, the decrease in the remaining supercoiled and open circular DNA in the powders during storage was much slower than that in solutions. In addition, the powders had higher transfection potency than the solutions containing the same amount of DNA. The effect of chitosan on the stability of DNA in solutions was not obvious in the solutions but it improved the stability of DNA in powders during manufacturing and storage. Thus, a gene powder with a cationic vector is a promising ready-to-use formulation for inhalation therapy of pulmonary diseases.

Synthesis of cross-linked circular DNAs using Hüisgen reaction.

Triazole-cross-linked ODNs were synthesized using Hüisgen reaction with 21 mer hairpin DNA possessing N-3 azidoethylthymidine and N-3 propargylthymidine at the 3' and 5'-terminals. Newly synthesized ODNs revealed thermally stable and their structures nearly retained those of non-cross-liked ODNs. This strategy is quite effective to prepare cross-linked circular ODNs.

Synthesis and characterization of topologically linked single-stranded DNA rings.

We investigated the synthesis of linked-ring DNAs by two DNA-ligation-based methods. In the first method, two DNA oligonucleotides were associated through a duplex segment of more than a full helical turn. Circularization of the entwined oligonucleotides by T4 DNA ligase resulted in two linked-ring DNAs with a total yield of approximately 40%. In the second method, a DNA oligonucleotide was circularized over a circular DNA template, resulting in the formation of approximately 10% linked-ring product. The circular nature of linked-ring DNAs was verified with exonuclease digestion and the existence of topological linkages was demonstrated by analyzing the electrophoretic mobility pattern of DNA products obtained from the digestion of each linked-ring DNA using specific restriction endonucleases. A linked-ring DNA library in which one of the two rings contained random-sequence nucleotides was also constructed and tested for compatibility with in vitro selection.

Advantages of the circular dumbbell decoy in gene therapy and studies of gene regulation.

Decoy oligodeoxynucleotides (ODN) that can reduce the trans-activity of transcription factors may be highly useful in gene therapy and the study of transcriptional regulation. Several different types of these double-stranded DNA decoys have been developed, including unmodified oligonucleotide duplexes, alphabeta-anomeric oligonucleotides, and oligonucleotide duplexes with methylphosphonate- and phosphorothioate-modified linkages. The latter ODNs have been particularly extensively studied but suffer from a number of limitations, including their insensitivity to polymerases, their lack of sequence specificity, and their tendency to activate immune responses. To resolve these problems, circular dumbbell (CD) double-stranded ODNs were developed. These CD ODNs are constructed by the circularization of the 3' and 5' ends of the oligonucleotides and enzymatic ligation. They exhibit high resistance to nucleases, are easily taken up by cells, and have a nontoxic unmodified backbone that resembles natural DNA. In this article, we review the method of constructing CD ODNs and their advantages compared to other modified ODNs for use as transcription decoys.

Head-to-tail oxime cyclization of oligodeoxynucleotides for the efficient synthesis of circular DNA analogues.

A convenient strategy for the synthesis of the analogue of cyclic oligodeoxyribonucleotides is presented. The cyclization of the oligonucleotide was accomplished through intramolecular oxime bond formation between a 5'-oxyamine moiety and a 3'-aldehydic group.

Statistical mechanics of sequence-dependent circular DNA and its application for DNA cyclization.

DNA cyclization is potentially the most powerful approach for systematic quantitation of sequence-dependent DNA bending and flexibility. We extend the statistical mechanics of the homogeneous DNA circle to a model that considers discrete basepairs, thus allowing for inhomogeneity, and apply the model to analysis of DNA cyclization. The theory starts from an iterative search for the minimum energy configuration of circular DNA. Thermodynamic quantities such as the J factor, which is essentially the ratio of the partition functions of circular and linear forms, are evaluated by integrating the thermal fluctuations around the configuration under harmonic approximation. Accurate analytic expressions are obtained for equilibrium configurations of homogeneous circular DNA with and without bending anisotropy. J factors for both homogeneous and inhomogeneous DNA are evaluated. Effects of curvature, helical repeat, and bending and torsional flexibility in DNA cyclization are analyzed in detail, revealing that DNA cyclization can detect as little as one degree of curvature and a few percent change in flexibility. J factors calculated by our new approach are well consistent with Monte Carlo simulations, whereas the new theory has much greater efficiency in computations. Simulation of experimental results has been demonstrated.

Artificial human telomeres from DNA nanocircle templates.

Human telomerase is a reverse-transcriptase enzyme that synthesizes the multikilobase repeating hexamer telomere sequence (TTAGGG)n at the ends of chromosomes. Here we describe a designed approach to mimicry of telomerase, in which synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes. Results show that the combination of a nanocircle and a DNA polymerase gives a positive telomere-repeat amplification protocol assay result for telomerase activity, and similar to the natural enzyme, it is inhibited by a known telomerase inhibitor. We show that artificial telomeres can be engineered on human chromosomes by this approach. This strategy allows for the preparation of synthetic telomeres for biological and structural study of telomeres and proteins that interact with them, and it raises the possibility of telomere engineering in cells without expression of telomerase itself. Finally, the results provide direct physical support for a recently proposed rolling-circle mechanism for telomerase-independent telomere elongation.