Topoisomerase-Based Preparation and AFM Imaging of Multi

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Topoisomerase-based preparation and AFM imaging of multi-interlocked circular DNA Tevin Li, Hao Zhang, lianzhe hu, and Fangwei Shao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00606 • Publication Date (Web): 08 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

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Bioconjugate Chemistry

Topoisomerase-based preparation and AFM imaging of multi-interlocked circular DNA Tevin Li†, Hao Zhang‡, Lianzhe Hu‡, and Fangwei Shao‡* †

‡

Lexington High School, 251 Waltham Street, Lexington, MA, 02421, USA

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore, 637371.

*Address correspondence to: [email protected] Tel:

+65 6592-2511

Fax:

+65 6791-1961

KEYWORDS: Interlocked DNA, DNA catenane, DNA nanotechnology, Topoisomerase, Kinetoplast DNA

ACS Paragon Plus Environment

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ABSTRACT

Multi-interlocked circular DNA structures have been highly demanded for fabricating complicate functional DNA architectures and nanodevices such as molecular switches, shuttles and motors. Even though various innovative methods have been developed in the past, creation of multiinterlocked circular DNA structures with defined numbers of DNA molecules and linking patterns are still challenging tasks nowadays. Here, we propose a top-down decatenation of kinetoplast DNA as a new approach for creating multi-interlocked circular DNA structures. Through optimizing the amount and reaction time of topoisomerase II, we synthesized completely mutually interlocked tri-circular, tetra-circular and oligo-circular DNA structures, which have not yet been acquirable through any other existing synthetic means. The catenation structures of multiple circular DNA were further verified through atomic force microscopic analysis of the backbone overlapping patterns and the circumference. It accordingly is our expectation that the top-down enzymatic approaches could offer highly interlocked network with defined numbers of circular DNA with simple protocols, and could consequently be beneficial to the design and fabrication of sophisticated functional molecules and nanodevices in the areas of supramolecular chemistry, DNA nanotechnology and material science.

INTRODUCTION

Interlocked DNA structures are topological collections of two or more intertwined circular DNA molecules, within which chemical intersections are absent between each other.1-2 These unique structural assemblies have been known to play crucial roles in constructing highly complex molecular architectures, such as Borromean rings and sophisticated knots, as well as for fabricating functional nanodevices such as molecular switches, shuttles and motors.3-8


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Consequently, development of methodologies for preparing diversified categories of interlocked DNA structures has drawn a great attention in the past decade.3,

9-16

Synthesis of non-

symmetrical catenane assemblies, for example, has been accomplished very recently through a strategy that

took advantage of curved conformation of A/T-rich tracts and DNA

complementarity principles.17 In addition, structurally predefined DNA catenanes were successfully constructed through utilization of specific inter-strand binding of polyamide molecules to certain particular sites of duplex DNA.16 Moreover, it was illustrated that two catenated DNA circles of different sizes could be prepared upon recombination reactions on circular toroidal duplex DNA structures.18 These previously reported methods were mostly bottom-up approaches for assembling interlocked DNA structures from smaller DNA segments. 3-18

Though bottom-up methods can provide wide diversity of catenane structures with different

building blocks, the methods often suffer from low yield, complicate multistep assembly protocol and high cost on preparing DNA building blocks.19 To circumvent these issues, we attempted to acquire interlocked DNA structures through top-down approach by disassembling a massive interlocked DNA networks. Here we report a simple one-step preparation of [2]-, [3]-, [4]catenanes ([n] is the number of circular DNA) and other multi-interlocked circular DNA structures from kinetoplast DNA through maneuvering the time and amounts of topoisomerase II (topo II), a type II topoisomerase from Homo sapiens. These interlocked DNA structures were further confirmed through electrophoretic analysis and atomic force microscopy. Among the newly obtained catenated structures, completely mutual-interlocked tri-circular and tetra-circular DNA display multiple interwining patterns that could be difficult to acquire through any other existing bottom-up synthetic means.

RESULTS AND DISCUSSION


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Kinetoplast DNA (Structure 1 in Fig. 1) is the mitochondrial DNA of trypanosomatids that consists of predominately ~5000 amassed minicircles.20 These minicircles contain approximately 2,500 base pairs each and are innately interlocked among themselves to form a massive and complex DNA network (Fig. 2B).21 Topo II is, on the other hand, a type II topoisomerase that can decatenate interlocked circular DNA by binding the cross site of two interlocked circular DNA and breaking one duplex DNA, facilitating the passage of the second circle through duplex break and ligating the cutting site to resume the original circular DNA structures (Fig. S1).22-24 When kinetoplast DNA (kDNA) is used as the substrate, each enzymatic action of topo II will detangle one interlock between two DNA minicircles at a time, which accordingly leads to the change of linking number by 2.25 It was reported previously that topo II was capable of transforming kinetoplast DNA into single DNA circle through its decatenation actions (Fig. 1A).26 Based on these previous observations, we speculated that if a massive network of kDNA could be decatenated in a controllable manner, intermediate structures between intact kinetoplast DNA and single DNA minicircles (e.g. [2]-, [3]-, [4]catenanes and other multi-interlocked minicircles) might be detainable and identifiable. Fig. 1B accordingly depicted our design of stepwise decatenation reactions of kinetoplast DNA to various catenane DNA.


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Figure 1. Decatenation of kinetoplast DNA. (A) Illustration of complete decatenation reactions of kinetoplast DNA by topo II under typical decatenation conditions;24 (B) Our envisaged stepwise decatenation of kinetoplast DNA through manipulation of amount of topo II. In Fig S2, intact kDNA (lane 2) showed no mobility on agarose gel, because the size of kDNA is extremely large (combination of ~5,000 DNA minicircles), which prevented it from passing through the pores of agarose gel networks. Further AFM examination revealed that the intact kDNA are a massive network of DNA minicircles and the backbone structures of individual DNA circles cannot be always well defined under AFM (Fig. 2B). In Fig S2, electrophoresis of enzymatic products of kDNA showed multiple bands with faster mobilities than intact kDNA (lane 3-6). The newly emerging bands after Topo II treatment presumably could be the intermediate products of decatenated kDNA, which could have smaller 3D size and pass agarose gel with much faster speeds than intact kDNA. The band intensity of these intermediates


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achieved maximum at 20 minutes of enzymatic reaction, which were used for the rest screening of topo II catalyzed decatenation in the paper.

Figure 2. Electrophoretic analysis and AFM images of kDNA and the complete decatenation products. (A) Electrophoretic analysis of decatenation products of kinetoplast DNA upon varying amount of topo II (lane 1, 1 kb DNA ladder; lane 2, kDNA; lane 3-7, decatenation of kDNA in the presence of 0.5 U (lane 3), 0.2 U (lane 4), 0.15 U (lane 5), 0.1 U (lane 6) or 0.05 U (lane 7) of topo II). (B) AFM images of intact kinetoplast DNA in lane 2. (C) AFM image of DNA products


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(measured circumferences = ~820 nm) obtained upon complete decatenation of kDNA by topo II in lane 3. (D) AFM image of DNA products that displayed no mobility shift (band 6 in lane 7). (E) AFM image of DNA products (measured circumferences = ~833 nm) that displayed the fastest mobility shift (band 1 in lane 7). In order to further optimize the yields of intermediate products of enzymatic decatenation of kDNA, the units of topo II used in the protocol were screened. When kDNA was incubated with topo II under typical literature-reported decatenation conditions (lane 3 in Fig 2A),23,24 kDNA is supposed to be completely distangled and a single new band with faster mobility shift were observed. AFM examination confirmed that the products in this band were single circular DNA (Fig. 2B). The measured circumferences of these circles in AFM images are around 820 nm, which matches that of calculated value of single DNA minicircles in kinetoplast DNA (~2,500 base pairs x 0.34 nm/base pair = ~850 nm). Furthermore, different units of topo II were applied to the decatenation reaction of kDNA (Fig 2A). Regardless of the amounts of topo II, every enzymatic treatment generated a fast moving band (band 1) with the same mobility as the band in lane 3. AFM examination confirmed that DNA products in the band were single DNA circles with circumferences around ~833 nm (Fig 2E), and the same final products of decatenated kDNA as those in lane 3 (circumferences = ~820 nm). By reducing topo II from 0.5 units to one tenth as listed in literature 0.05 units (lane 4 to lane 7 in Fig. 2A), the yield percentages of intermediate products, band 2 to band 5 increased significantly to ~40% of single circular product (Fig S3). Whereas after band 6 in lane 7 of Fig. 2A was isolated, AFM studies in Fig 2D showed that DNA in band 6 though displayed no mobility shift during electrophoresis, was different to the intact kDNA. That is, the DNA product in band 6 had the overall size significantly smaller that of intact kDNA and the interlocked network was much less


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complicated, which was already the partial decatenation products of kDNA, though it was still difficult to identify the numbers and interlocking patterns of minicircles. Besides band 1 and band 6, the intermediate products in the second fastest moving band (band 2 in lane 7 of Fig 2A) were purified and were identified as two interlocked DNA minicircles by AFM imaging (Fig. 3A1). The circumferences of the two interlocked DNA circles under AFM are nearly equal (~838 nm and ~823 nm respectively, Fig. 3A2), which is evident that the two interlocked DNA circles were originated from kinetoplast DNA. The two cross sites between the two circles can be clearly visualized under AFM (Fig 3A2). The interlocked patterns between the two circular rings can be determined by measuring the heights of DNA contours at the two cross sections as shown in Fig 3A3. When one ring resided on top of the other one at cross section, eg cross 1 annotated in Fig 3A2, the heights of cross can be measured along DNA contours of two rings, along either blue ring strands or red ring strands (blue and red arrows in Fig 3A2). DNA heights would have a graduate climbing up if AFM tip was moving along the ring strands on the top. On the contrary, a sudden rising up in sample height would be observed if AFM scan is along DNA ring at the bottom of the cross section. Steeper rising slope and narrower peak width were observed for both height analysis of DNA contours lying at bottom of the cross 1 and 2 in Fig 3A3. Hence it can be deduced that blue ring lied below red ring at cross 1 and the opposite situation occurred at cross 2, which evidently showed the two minicircles interlocked to form [2]catenane. By analyzing the section heights from two DNA contours, the scheme of interlock patterns in [2]-, [3]catenanes can be drawn as shown in Fig 3A5 and 3B3.


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Figure 3. AFM examination of multi-interlocked DNA circles that are corresponding to the products in band 2, band 3, band 4 and band 5 in lane 7 of Fig. 2A. (A) AFM images of interlocked dual circular DNA (A1) are isolated from band 2. Two DNA circles shown in A2 are interlocked (section analysis in A3, 3D image in A4 and scheme in A5). (B) AFM images of band 3 (B1) contains two interlocking pattern (B2, B4). Schemes (B3, B5) and section analysis (B6) of these tri-interlocked DNA structures are shown, respectively; (C) AFM image (C1) of band 4 and schemes (C2, C3) of interlocked tetra-circles; (D) AFM image of the smear band 5. In addition to the [2]catenane, the DNA products in the third fastest moving band (band 3) in lane 7 in Fig. 2A turned out to be [3]catenanes (Fig 3B). Our 3D AFM analysis revealed that


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there were in fact two interlocking types of [3]catenanes. In the first type of [3]catenanes, the three DNA circles, like in celtic knots, are completely mutually catenated with each other (Fig. 3B2). In other words, each DNA circle is interlocked twice with other two DNA circles, which make the number of crossings within this molecular assembly equal to 6 (2+2+2). To the best of our knowledge, this type of completely mutually interlocked [3]catenanes has not yet been attainable by any other synthetic means. The second type of [3]catenanes (Fig. 3B4) are only interlocked twice among themselves (partially mutually interlocked), the crossing number of which should be assigned as 4 (2+2). More specifically, as illustrated in Fig. 3B5, both circular DNA 1 (blue) and circular DNA 2 (green) are interlocked with circular DNA 3 (red) while there is absent of interlock between circular DNA 1 (blue) and circular DNA 2 (green). It is our speculation that one of the pathways that generated the chain type tri-circles would be one more step of enzymatic decatenation on celtic knot type of tri-interlocked DNA during the treatment of topoisomerase II on kinetoplast DNA (Fig. S4). In order to know whether other types of multi-interlocked DNA had been produced in our designed decatenation reactions, the DNA products in band 4 in lane 7 in Fig. 2A were isolated. As shown in Fig. 3C, the products turned out to be [4]catenanes with various interlocking topologies. Because the tetrameric circular DNA assemblies possess much higher molecular weight and more complex catenated structures, the backbone patterns in the AFM images were too overlapped to be clearly identifiable as those of [2]- and [3]catenanes. We can only tentatively draw the schemes of possible interlocking topologies of several tetra-circles in Fig. 3C2 and 3C3 to demonstrate the complication of the catenation network on the level of four minicircles. Furthermore, a fraction of the smear bands above band 4 in lane 7 of Fig. 2A was subsequently purified. Our AFM examination revealed that the DNA products in the smear bands


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are interlocked structures of oligomeric DNA circles (oligo-interlocked DNA) (Fig. 3D). Due to their structural complexity and the sensitivity limitation of AFM, the number of circular DNA molecules in the interlocked structures shown in Fig. 3D cannot be determined precisely. However, it was apparent that there were more 10 molecules of DNA circles catenated together in the oligo-interlocked DNA (Fig. 3D) though the number of minicircles may not be identical in different entities. Since oligo-interlocked DNA entities were clearly identifiable from decatenation of kinetoplast DNA (Fig. 1B), we would envisage that penta-, hexa-, hepta- and octa-interlocked DNA structures could be achievable as well if partial decatenation would be carried out on the oligomeric assemblies through further precisely controlling the amount of topo II (Fig. S5).

CONCLUSION

The processes of decatenation of kinetoplast DNA was well controlled through adjusting the amount of topo II and the time of treatment. Our electrophoretic analysis and AFM examination showed that interlocked [2]-, [3]catenanes and other oligo-interlocked DNA as intermediate products of kDNA decatenation are clearly identifiable. These observations suggest that topo II disentangles the interlocking network in kDNA into smaller and less complicate entities following a uncoordinated fashions. Most interestingly, two different types of [3]catenanes, completely mutually interlocked and chain type, were obtained by our protocol. Owing to the topological complexity, the completely mutually interlocked DNA (Fig. 3B2) has not yet been attainable via any existing synthetic methods. Using our protocol here, multiple circular DNA with mutual interlocking structure can be readily prepared by one step enzymatic reaction and


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could consequently be beneficial to the design and fabrication of complicate functional biomolecules and nanodevices in the future. ASSOCIATED CONTENT Supporting Information. Detailed experimental procedures, scheme for enzymatic reactions, the effect of reaction time, the ratio of different products, and AFM images. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors thank the financial support by NTU Nanyang Assistant Professorship (M4080531) and Singapore MOE Tier 2 grant (M4020163). REFERENCES 1. 2. 3.

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Topoisomerase-Based Preparation and AFM Imaging of Multi

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