Single-stranded DNA Rings Research Articles

Robust Storage of Chinese Language in a Pool of Small Single-Stranded DNA Rings and Its Facile Reading-Out

Abstract Archival storage in DNA is one of the most challenging themes in rapidly growing information technology. In addition, its practical applications are more difficult due to complicated data analysis, instability of long and linear DNA strands (>1000 nt), and other factors. In the present study, we have developed a simple and eminent DNA-based storage system in which small DNA rings are employed as memory units. Compared with previous approaches, this methodology has advantages of robustness, low cost, convenience and so on. In high density, Chinese language was directly stored in a pool of 76-nt-long single-stranded DNA rings (designated as “Info-Store”), in which each ring memorized the index number and five Chinese characters (or marks). During “Read-Out”, all the ssDNA rings in the pool were simultaneously amplified by hyper-branched rolling cycle amplification (HRCA), and their sequences were accurately determined by a portable MinION sequencer aided by a personal computer. Then, the original Chinese text was precisely and smoothly decoded by simple data analysis.

Two-Holder Strategy for Efficient and Selective Synthesis of Lk 1 ssDNA Catenane

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it governs the feasibility of dynamic motions. However, there has been no established way to synthesize catenanes in which Lk is controlled to a predetermined value. This paper reports a new methodology to selectively synthesize Lk 1 catenanes composed of single-stranded DNA rings, in which these rings can most freely rotate each other due to minimal inter-ring interactions. To the mixture for the synthesis, two holder strands (oligonucleotides of 18–46 nt) were added, and the structure of the quasi-catenane intermediate was interlocked through Watson–Crick base pairings into a favorable conformation for Lk 1 catenation. The length of the complementary part between the two quasi-rings was kept at 10 bp or shorter. Under these steric constraints, two quasi-rings were cyclized with the use of T4 DNA ligase. By this simple procedure, the formation of undesired topoisomers (Lk ≥ 2) was almost completely inhibited, and Lk 1 catenane was selectively prepared in high yield up to 70 mole%. These Lk 1 catenanes have high potentials as dynamic parts for versatile DNA architectures.

Cyclization of secondarily structured oligonucleotides to single-stranded rings by using Taq DNA ligase at high temperatures.

Single-stranded DNA rings play important roles in nanoarchitectures, molecular machines, DNA detection, etc. Although T4 DNA ligase has been widely employed to cyclize single-stranded oligonucleotides into rings, the cyclization efficiency is very low when the oligonucleotides (l-DNAs) take complicated secondary structures at ambient temperatures. In the present study, this problem has been solved by using Thermus aquaticus DNA ligase (Taq DNA ligase) at higher temperatures (65 and 70 °C) where the secondary structures are less stable or completely destroyed. This method is based on our new finding that this ligase successfully functions even when the splint strand is short and forms no stable duplex with l-DNA (at least in the absence of the enzyme). In order to increase the efficiency of cyclization, various operation factors (lengths and sequences of splint, as well as the size of the DNA ring) have been investigated. Based on these results, DNA rings have been successfully synthesized from secondarily structured oligonucleotides in high yields and high selectivity. The present methodology is applicable to the preparation of versatile DNA rings involving complicated secondary structures, which should show novel properties and greatly widen the scope of DNA-based nanotechnology.

Highly efficient preparation of single-stranded DNA rings by T4 ligase at abnormally low Mg(II) concentration.

Preparation of large amount of single-stranded circular DNA in high selectivity is crucial for further developments of nanotechnology and other DNA sciences. Herein, a simple but practically useful methodology to prepare DNA rings has been presented. One of the essential factors is to use highly diluted T4 ligase buffer for ligase reactions. This strategy is based on our unexpected finding that, in diluted T4 buffers, intermolecular polymerization of DNA fragments is greatly suppressed with respect to their intramolecular cyclization. This promotion of cyclization is attributable to abnormally low concentration of Mg2+ ion (0.5–1.0 mM) but not ATP in the media for T4 ligase reactions. The second essential factor is to add DNA substrate intermittently to the mixture and maintain its temporal concentration low. By combining these two factors, single-stranded DNA rings of various sizes (31–74 nt) were obtained in high selectivity (89 mol% for 66-nt DNA) and in satisfactorily high productivity (∼0.2 mg/ml). A linear 72-nt DNA was converted to the corresponding DNA ring in nearly 100% selectivity. The superiority of this new method was further substantiated by the fact that small-sized DNA rings (31–42 nt), which were otherwise hardly obtainable, were successfully prepared in reasonable yields.

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 ∼40%. In the second method, a DNA oligonucleotide was circularized over a circular DNA template, resulting in the formation of ∼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.

E. coli and M. luteus DNA topoisomerase I can catalyze catenation or decatenation of double-stranded DNA rings

Escherichia coli and Micrococcus luteus DNA topoisomerase I are found to promote catenation of double-stranded DNA rings. At low DNA concentration dimeric catenanes are the major catenated products; at high DNA concentration or when spermidine is present, catenanes containing more than two rings are formed. There is no requirement of extensive sequence homology between the component rings forming a catenane; dimeric catenanes between Pseudomonas phage PM2 DNA and E. coli plasmid pBR322 are readily formed. The formation of a dimeric catenane by these type I topoisomerases, however, requires the presence of at least one preexisting single-chain scission in one of the two component rings. This is in contrast to the cases with the type II DNA topoisomerases which can form catenanes made of covalently closed rings only. The catenanes formed by the type I enzymes can be unlinked by the same enzymes, or by DNA gyrase, a type II enzyme, upon dilution of the isolated catenanes. The catenation and decatenation of duplex DNA rings adds a fourth type of reaction promoted by these type I DNA topoisomerases to the three reported previously: relaxation of superhelical DNA, interconversion between single-stranded DNA rings with and without knots and the intertwining of single-stranded DNA rings of complementary sequences into a covalently closed duplex ring with a high linking number. All four topoisomerization reactions involve the crossing of one DNA strand through a transient break of another DNA strand. The new reaction reported here suggests that such a crossover event might not require pairing of complementary nucleotide sequences.

Escherichia coli DNA topoisomerase I catalyzed linking of single-stranded rings of complementary base sequences

Eco DNA topoisomerase I (E. coli omega protein) has been observed to catalyze the formation of double-stranded, covalently closed DNA from complementary single-stranded DNA rings, a novel reaction which is topologically forbidden without the enzyme-catalyzed breakage and rejoining of DNA backbone bonds. Incubation of a mixture of single-stranded PM2 DNA rings of complementary base sequences with omega yields a species with a sedimentation coefficient in an alkaline medium characteristic of a covalently closed circular double-stranded DNA. Buoyant density measurements in CsCl at alkaline pH also identify the product as a covalently closed duplex ring. If the omega-catalyzed reaction is stopped short of completion, highly negatively supercoiled molecules are formed which sediment more slowly in an alkaline medium than the final duplex product. As the reaction proceeds the mean sedimentation rate of the intermediates increases. This is in agreement with the expectation that the linking number between the two complementary rings increases gradually during the course of the reaction from zero to that of a relaxed covalently closed circular DNA duplex. The possible role of DNA topoisomerases in genetic recombination is discussed.

Knotted single-stranded DNA rings: A novel topological isomer of circular single-stranded DNA formed by treatment with Escherichia coli ω protein

Treatment of single-stranded circular phage fd DNA with Escherichia coli ω protein yields a new species which sediments 1.2 to 1.5 times faster than the untreated DNA in an alkaline medium. The infectivity of this species in spheroplast assays, after purification of the DNA by zone sedimentation in an alkaline sucrose gradient, is only slightly lower than that of untreated fd DNA. The formation of this species requires Mg(II) and is strongly dependent on salt concentration and temperature. At 37 °C, over 85% of the input DNA can be converted to this form when incubation is carried out in media containing 0.15 to 0.25 m-salt. The yield decreases with increasing temperature or decreasing salt concentration. The increase in sedimentation coefficient of fd DNA in an alkaline medium following treatment with ω is not due to protein binding, as no change was observed upon treatment of the product with phenol or Pronase. Furthermore, neither the buoyant density of this new species in neutral CsCl nor its sedimentation coefficient in a neutral medium is significantly different from the corresponding properties of untreated fd DNA. Examination by electron microscopy shows that the new form has the appearance of a knotted ring of about the same contour length as an untreated monomeric single-stranded fd DNA. The new form can be converted to full-length linear fd DNA by treatment with pancreatic DNAase I. The rate of conversion is approximately the same as that of untreated circular fd DNA to the linear form. These results show that the new form of fd DNA is a novel topological isomer: a knotted single-stranded DNA ring. It is also found that further treatment of the knotted DNA rings with ω at low ionic strength can reverse the reaction, i.e. the knotted DNA rings can be converted back to simple DNA rings indistinguishable from fd DNA from the phage. At intermediate ionic strength the two forms are interconvertible and form an equilibrium mixture. Results similar to those obtained for fd DNA have also been observed for single-stranded circular φX174 DNA. A mechanism based on the known activity of ω protein on double-stranded DNA, the secondary structure of a single-stranded circular DNA, and the experimental results described here is proposed.

The process of infection with bacteriophage ΦX174 : VII. Ultracentrifugal analysis of the replicative form

A procedure is described for the preparation of a purified ΦX174 replicative form DNA. Ultracentrifugal analyses, zone sedimentation and density gradient of this DNA, both when native and when denatured, are described. At neutral pH, two major sedimentation components in the purified replicative form of 21·2 s (I) and 16·2 s (II) (as the sodium salts) are observed. At alkaline pH, in high ionic strength medium, or at neutral pH in formamide, (I) gives rise primarily to a rapidly sedimenting component, the denatured double-stranded DNA ring. Under the same conditions, (II) gives rise to infective single-stranded DNA rings. It is concluded that (I) is the closed double-strand DNA ring, whereas (II) is composed of double-stranded DNA rings in which one strand is open. Upon sedimentation at neutral pH of an unfractionated nucleic acid extract of ΦX-infeeted cells (in chloramphenicol) and analysis of the sedimentation distribution of infectivity, component (I) is the major infective substance observed. When an alkali-denatured replicative form preparation is sedimented in an alkaline sucrose gradient at low ionic strength at pH 11, three infective components of S = 33, 23 and 14 s are observed. Component (I) gives rise to both the 33 s and 23 s components, whereas (II) gives rise only to the 14 s single-strand rings. The 33 s and 23 s components are the denatured and native or renatured double-stranded rings, respectively. These are present in variable proportions, dependent upon the circumstances of denaturation. Comparisons of infectivity indicate that the denatured double-stranded DNA is 25 times as infective as the native form in the assay system employed. Upon equilibrium density-gradient sedimentation in alkali of the purified ΦX replicative form DNA, four infective components are observed, with buoyant densities of 1·78 (ρ-1), 1·765 (ρ-2), 1·76 (ρ-3) and 1·75 (ρ-4) g/ml. ρ-1 is the denatured double-stranded DNA ring and corresponds to the rapidly sedimenting component in velocity sedimentation, ρ-2 is the viral DNA. ρ-3 is believed to be double-stranded, and ρ-4 appears to be single-stranded. Component (I) of the replicative form which gives rise to ρ-1 denatures at a higher pH than that (component II) which gives rise to the viral DNA.