Article pubs.acs.org/Macromolecules
Bridged DNA Circles: A New Model System To Study DNA Topology Alexander Vologodskii* Department of Chemistry, New York University, New York, New York 10003, United States S Supporting Information *
ABSTRACT: We demonstrate the assembly of two bridged circular DNA molecules. The circles are specially engineered plasmid DNAs a few thousands base pairs in length connected by the synthetic double-stranded linker. The construct represents a very convenient system for studying DNA topology and its enzymatic transformations, since at thermodynamic equilibrium the circles have comparable probability to adopt two different topological states and the exchange between the states is an intramolecular reaction. We obtained the equilibrium distribution of these states, in which the circles form the simplest link or remain unlinked, by opening and closing the long sticky ends made in one of the bridged circles. Separation of the linked and unlinked circles by gel electrophoresis allowed us to determine the fraction of linked circles. The obtained value is in very good agreement with the results of computer simulation.
I. INTRODUCTION Circular polymer chains can have different topologythey can be unknotted or form various knots as well as various topological links with other circular chains.1−3 These topological properties were the subject of many theoretical and experimental studies. Topology is especially important for circular DNA, and nearly all experimental studies of topological properties were performed on these molecules. To solve topological problems in DNA molecules, nature developed special enzymes, DNA topoisomerases, which can change DNA topology.4 Mechanisms of topological transformations catalyzed by these enzymes are the subject of numerous biochemical and biophysical studies (see refs 5−16 as examples). It was discovered that a family of these enzymes, type IIA DNA topoisomerases, are capable to dramatically reduce the fractions of linked and knotted circular DNA relative to the corresponding fractions at thermodynamic equilibrium.17 Although this puzzling discovery attracted a lot of attention over the years,18−24 we do not yet know how the enzymes solve this task. A good understanding of topological transformations in circular DNA molecules is important for our general understanding of DNA functioning inside living cells. To study DNA topological properties, we need a convenient model system. The usual systems based on DNA knots or links (catenanes) have fundamental limitations. The equilibrium fraction of knots in DNA molecules a few kb in length, mainly used in the studies, is very low.25,26 Experimental measurements and theoretical investigations of such small fractions are difficult. The equilibrium fraction of catenanes depends on DNA concentration in solution and therefore can be shifted to a range convenient for the measurements.27 However, catenation is an intermolecular reaction, and as such, it is much more difficult for theoretical analysis.28 An ideal object for the analysis of DNA topological transformations is a single DNA chain that can adopt at least two different topological states with comparable probability. In this paper we describe © 2012 American Chemical Society
the preparation of a new object with both of the above properties. The object consists of two circular DNA molecules a few kb in length linked by a short DNA bridge. A system of bridged circular chains has been used as a convenient theoretical model for the linking/unlinking of circular DNA molecules.29 However, for quantitative analysis of a phenomenon we need to use the same objects in experimental measurements and theoretical analysis. In this paper equilibrium topological properties of the bridged DNA circles are investigated both experimentally and computationally.
II. PREPARATION OF BRIDGED DNA CIRCLES The constructs of the two bridged circular DNAs were assembled from two different plasmids and a synthetic connector. The plasmids are derivatives of pUC19 DNA with inserts of 55 and 73 bp in length, which contain the sequence elements needed for the assembly and successive experiments. (The sequences of the inserts and the synthetic linker are given in the Supporting Information.) Thus, the two circular DNA molecules used for the assembly consist of 2735 and 2753 bp. To obtain the plasmids, DNA pUC19 was treated by restriction endonucleases Pst I and Hind III to convert the circular DNA into linear form with the corresponding sticky ends. Then the restriction enzymes and the short segment between their recognition sites were removed from the solution by applying the PCR purification procedure (QIAGEN). Two different plasmids were obtained by adding different synthetic inserts with the corresponding sticky ends to the linearized plasmid. After ligation the mixtures and cloning the plasmids, two desired circular DNA molecules were obtained by cloning the plasmids into E. coli cells and extracting the plasmids from the grown cultures. The sequences of the obtained plasmids were checked by direct sequencing (GENEWIZ). Each insert has a removable single-stranded segment of 28 bp in length (the segments had different sequences). The single-stranded Received: March 13, 2012 Revised: April 18, 2012 Published: April 30, 2012 4333
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nicks needed for the segment removal were produced by the sequencespecific nicking endonucleases, Nt.BspQ1 and Nb.BsrD1 (New England Biolabs, NEB). The recognition sites for these endonucleases were part of the insets. Very low GC content of the removable segments allowed for them to melt out before the melting of the rest of the DNA. The melted out oligos were then hybridized with a large excess of complementary oligos followed by the removal of all oligos from the solution by applying the PCR purification procedure. The plasmids and the connecting synthetic oligonucleotide linker were mixed in the molar ratio 1:1:1. The connector has a double-stranded region of 48 bp and single-stranded ends whose sequences are
transformations in the bridged circular molecules. Indeed, all possible topological forms of the bridged plasmids are well separated from the isolated circles.
III. EQUILIBRIUM DISTRIBUTION OF TOPOLOGICAL STATES OF THE BRIDGED CIRCLES a. Experimental Results. The bridged DNAs can be in different topological states, first of all in the states where the circles are unlinked and form the simplest link, 21 (see Figure 4). Of course, initially all the assembled bridged circles are unlinked, and to obtain the equilibrium distribution of topological states, we need to temporarily convert one circle of each pair into a linear form. This can be done by opening and closing the long sticky ends of one of the circles. The closing of sticky ends is a very slow process that creates equilibrium distribution of topological states.25−27,30 To make sticky ends in one of the bridged circles, two recognition sites for another nicking enzyme, Nb.BsmI (NEB), were incorporated into the plasmid insert. So, treating the construct by the nicking endonuclease makes 20 nucleotide sticky ends. The second circle has a single recognition site for the same nicking enzyme to convert the closed circular form of the circle into a nicked form. After treating the construct by Nb.BsmI, the sample was purified by the PCR purification procedure (QIAGEN) and dissolved in solution containing 10 mM Tris and 0.5 mM EDTA (buffer I). First, we needed to investigate the opening and closing of the sticky ends. To achieve this goal, we needed a way to fix the fractions of open and closed forms of the circle at a desired moment of time. We did it by adding to the sample 10 μM of oligonucleotide complementary to one of the sticky ends, as was suggested earlier.25 After making the final salt concentration of 100 mM of NaCl and 10 mM of MgCl2 (buffer II), the oligonucleotide quickly hybridizes with the opened sticky end, preventing further cyclization. After that the fractions of the construct with open and closed sticky ends can be separated by agarose gel electrophoresis and quantified. Through this process we found that the sticky ends dissociate completely after 5 min of incubation in buffer I at 56 °C. The sticky end closing in buffer I at 37 °C requires more than 2 days, while in buffer II the half-time of closing at this temperature is around 50 min−1. With this knowledge we were able to create the equilibrium distribution of the topological states of the bridged circles. After the opening of the sticky ends in buffer I the construct was cooled to 37 °C, transferred to buffer II, and incubated for 5 h. The obtained topological forms of the bridged circles were separated by agarose gel electrophoresis. To identify electrophoretic bands corresponding to the most probable states of the construct, we made two additional samples. The sample with 100% of unlinked closed circles is obtained after nicking the construct by Nb.BsmI but not opening the sticky ends. The sample with a fraction of opened sticky ends was obtained by the partial reopening of the closed sticky ends and adding the complementary oligonucleotide. Comparison of the bands of all three samples allowed for unambiguous identification of all three of the most probable states of the construct (Figure 3). The gel was stained by SYBR Gold and quantified by scanning the band fluorescence on the Storm 860 Molecular Imager (GE Healthcare). We found that the fractions of unlinked and singly linked circles equal 0.76 ± 0.02 and 0.24 ± 0.02, respectively.
Figure 1. Assembling of bridged DNA circles. The gapped plasmids are hybridized with a synthetic DNA connector. The connector has a double-stranded region of 48 bp and single-stranded ends whose sequences are complementary to the gaps made in the plasmids. The single-stranded nicks in the assembled structure have to be ligated. complementary to the gaps made in the plasmids (Figure 1). It also has a unique restriction site for Cla I endonuclease used for the analysis of the assembled product. The connector was hybridized with the gapped circular DNAs, and the single-stranded nicks in the assembled construct were ligated. The electrophoretic analysis of the obtained products is shown in Figure 2. The photograph of the gel shown in Figure 2 demonstrates that the protocol leads to a good yield of the desired product. Although a substantial amount, about 50%, of the plasmids remain unbound, this is not an obstacle for quantitative measurements of topological
Figure 2. Electrophoretic separation of the ligation products obtained during the assembly of the bridged DNA circles. Upon nick ligation each plasmid obtains its own linking number, Lk. The topoisomers of the bridged circular DNAs with different values of Lk1 + Lk2 (lane 2) move as linear DNA markers of 4−7 kb. The individual plasmids are represented by only five topoisomers and nicked circles. The topoisomers of unbound plasmids move independently, and the corresponding sets of bands are slightly shifted relative to one another since the lengths of the plasmids differ by 18 bp. As expected, the bands that correspond to the bound circular DNAs disappear after the connector is cut by endonuclease Cla I (lane 3). 1.0% SeaKem LE agarose (Lonza) gel electrophoresis was run in TAE buffer at 5 V/cm for 5 h. 4334
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Figure 3. Gel electrophoresis of the nicked bridged circular DNAs. The three most probable states of the bridged circles are diagrammed on the right side of the figure. The construct equilibrated by opening and closing one of the circles was loaded in lane 3. The sticky ends of the sample in lane 4 have not been opened, and therefore the sample corresponds to the unlinked circles. Lane 2 contains all three of the most probable states of the construct including the state with the partially reopened circle (see the text). Thus, two bands in lane 3 of the gel correspond to the equilibrium fractions of the bridged DNAs with linked and unlinked circles. The distribution corresponds to near physiological ionic condition (buffer II). 1 kb Plus DNA ladder (Invitrogen) was loaded in lane 1. The different forms of the construct were separated by 1.1% SeaKem LE agarose (Lonza) gel electrophoresis in TAE buffer at 5 V/cm for 5 h. DNA bands were visualized by SYBR Gold (Invitrogen) staining.
Figure 4. Two typical simulated conformations of the bridged circular DNA molecules 2800 bp in length. The length of the bridge corresponds to 42 bp. It is assumed that each circular DNA has a single-stranded nick, so there is no torsional stress in the model chains. The circular DNAs at the top are unlinked while the circles at the bottom form the simplest link, 21. Colors are used for convenience to show different parts of the construct.
b. Computer Simulation. We also obtained the equilibrium distribution of topological states of bridged DNA circles by computer simulation. In the simulation DNA double helix was modeled as a discrete wormlike chain whose segments are rigid cylinders of a particular diameter.28,31 It is known that this simple model provides a very accurate description of DNA large-scale conformational properties.31 The length of the model segments is a computational parameter of the model which should be chosen to be sufficiently small so the property of interest does not change on further reduction of the segment length. In the current simulation the segment length corresponded to 4.4 nm. The model has two physical parameters: DNA persistence length, a, and the effective diameter of the chain, d (the diameter of the impenetrable cylinders). The energy of the model chain depends on the angles between the directions of adjacent segments. the corresponding bending rigidity constant is specified by the value of a.31 The value of d accounts for the electrostatic repulsion between the DNA segments and therefore strongly depends on the ionic conditions.32 For the conditions used in the experiment the values of a and d equal 44 and 4 nm, correspondingly.32−35 The Metropolis Monte Carlo procedure was used to sample the equilibrium conformational set of the bridged circles.28,31 During the procedure the chain segments can pass through one another, so topological state of the construct changes. To calculate the equilibrium distribution of the topological states, the topology of each conformation of the simulated set was determined by calculating the Alexander polynomial for two circular contours, Δ(s,t), for s = −1 and t = −1.28 Typical simulated conformations of the bridged circles are shown in Figure 4. The fractions of two types of links, 21 and 41, obtained in the simulations, are shown in Figure 5 as a function of the circle size. It is interesting that the fraction of links 21 weakly depends on the length of DNA circles. This is partially due to the fact that the fractions of links 41 and even more complex links
Figure 5. Equilibrium probability of links 21 and 41 for two bridged circular DNA molecules as a function of their length. The length of the connecting DNA segment is 42 bp. The open symbols correspond to the data obtained by Monte Carlo simulation of the equilibrium conformational ensemble of the system. The measured fraction of 21 links is shown by the filled triangle.
increase in the larger bridged circles. The measured fraction of 21 links is also shown in the figure. As we can see, the measured and simulated fractions of 21 links are in a very good agreement, although the way of detection used in the experiment did not allow us to observe 41 links whose fraction is small.
IV. CONCLUSIONS We designed and prepared a construct of two bridged DNA plasmids. Then we showed that equilibrium probability of forming link 21, determined experimentally and obtained by computer simulation, is high. Even for circular molecules of 3 kb in length, it is close to 0.25. This makes linking in the bridged circles much more convenient for quantitative analysis than formation of knots in the circular chains. The equilibrium fraction of the simplest knots, trefoils, in 6 kb DNA, in a diluted solution, is close to 0.01 under the same ionic conditions.25,26 This difference in the probabilities of trefoil knots and the 21 4335
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link is due to the fact that the link is essentially a simpler topological formation than the knots. It is illustrated by the fact that the 21 link can be drawn with only two crossings on a projection while trefoil has at least three crossing. On the other hand, all topological transformations in the bridged circles are intramolecular reactions, which is very important for their computational analysis. The described procedure of obtaining bridged DNA circles combines specially prepared plasmids grown inside the cells and synthetic DNA chains. We have shown that this combination of elements can be an efficient way of designing large DNA constructs that can serve a broad range of purposes in the booming area of DNA nanotechnology.
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ASSOCIATED CONTENT
S Supporting Information *
The sequences of synthetic oligonucleotides used in the study. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The author thanks Neville Kallenbach and Maria Vologodskaia for helpful discussions of this work.
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REFERENCES
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