Communication pubs.acs.org/Biomac
A Smart DNA Tetrahedron That Isothermally Assembles or Dissociates in Response to the Solution pH Value Changes Zhiyu Liu, Yingmei Li, Cheng Tian, and Chengde Mao* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States S Supporting Information *
ABSTRACT: This communication reports a DNA tetrahedron whose self-assembly is triggered by an acidic environment. The key element is the formation/dissociation of a short, cytosine (C)-containing, DNA triplex. As the solution pH value oscillates between 5.0 and 8.0, the DNA triplex will form and dissociate that, in turn, leads to assembly or disassembly of the DNA tetrahedron, which has been demonstrated by native polyacrylamide gel electrophoresis (PAGE). We believe that such environment-responsive behavior will be important for potential applications of DNA nanocages such as on-demand drug release.
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forms at pH 5.0 and dissociates at pH 8.0. In the current design, C+G-C triplets (pH sensitive) and TA-T triplets (not pH sensitive) each make up 50% of the triplex. In addition to the triplex-forming sticky ends, we also keep two bases that can form Watson−Crick base-pairing. While the triplex will provide the cohesion strength to hold the individual motif together, the Watson−Crick basepairs will direct the local geometry of the intermotif cohesion to ensure the overall polyhedral geometry. We followed reported protocol to assemble the pHresponsive DNA tetrahedron.26 Briefly, strands L, M, and S at a ratio of 1:3:3 were dissolved in a Mg2+ containing neutral buffer (pH 8.0) and slowly cooled down from 95 to 4 °C to form the individual DNA three-point-star motif. Then 1.0 M HCl was added to adjust the solution pH to 5.0 to trigger the triplex formation and the DNA tetrahedron assembly. After assembly, we analyzed the DNA complexes by native PAGE (Figure 2) because our past studies on DNA polyhedra proved that native PAGE is one of the most efficient methods to verify the formation of DNA polyhedra. In the current study, we also prepared a series of previously, thoroughly characterized size markers: (i) “individual motif” was a single three-point-star motif with blunt ends; and (ii) “motif-oligomers” contained a series of oligomers of three-point-star motif with sticky ends. Among them, the DNA tetrahedron (composed of four threepoint-star motifs) was the most abundant complex. 25a Comparing the native PAGE at pH 5.0 and 8.0, while most DNA samples had very similar electrophoretic mobilities, the DNA samples of L+M+S (1:3:3) differed dramatically under these two conditions. At pH 8.0, triplex could not form, thus, L +M+S (1:3:3) existed as an individual motif and migrated similar to the blunt-ended, individual three-point-star motif marker. In contrast, at pH 5.0, triplex readily formed and L+M +S (1:3:3) existed as DNA tetrahedron, appearing as a sharp band (with a yield of 60% as estimated by ImageJ, an image
NA is not merely the genetic material for life, but also an excellent building block for nanoconstruction.1−3 Various well-defined nanostructures have been assembled from DNA molecules. One potential application for DNA nanostructures is on-demand material delivery.4,5 The general idea is to use DNA nanocages to encapsulate cargos. With certain environmental cues, the DNA nanocages disassemble or change conformations, leading to cargo release.4,5 Along this line, DNA nanocages have been well developed,6−19 but the strategies for controllable disassembly/reconfiguration of DNA nanocages are seldom and relatively underdeveloped.4,5,20,21 Conceptually, DNA nanomachines,22−25 with the ability of reversible structural conversion, can perfectly perform those functions. Herein, we report a strategy to use one of the DNA nanomachine concepts to reversibly assemble/disassemble a DNA nanocage. The reported work here (Figure 1) is built on two lines of previous works: self-assembly of symmetric DNA nanocages26 and triplex-based, pH-responsive DNA nanomachines.27 Recently, we have developed a general approach to assemble well-defined DNA polyhedra from symmetric star-shaped nanomotifs.28 For instance, a tetrahedron can be assembled from four copies of identical three-point-star motifs. Each motif contains one long, 3-fold repetitive strand (L), three identical medium strands (M), and three identical short strands (S). At the peripheral ends of each branch of the motif, there are two complementary single-stranded overhangs (sticky ends). Watson−Crick base-pairing of sticky ends among the motifs brings motifs together to form DNA polyhedra. The key element of the current work is a C-containing, pH-sensitive DNA triplex.28 We have introduced triplex into the sticky end regions. In an acidic solution (e.g., pH 5.0), C is half protonated as C+, which can interact with a G-C basepair through Hoogsteen hydrogen bonding (H-bonding) to form C+G-C triplets.29 When the solution becomes neutral (e.g., pH 8.0), C+ losing a proton becomes C, which cannot form Hoogsteen Hbond with a normal G-C basepair. Hence, the C+G-C triplets dissociate into a C and a G-C basepair. Thus, a C-containing DNA triplex is sensitive to the solution acidity (pH value): © XXXX American Chemical Society
Received: March 25, 2013 Revised: April 24, 2013
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Figure 2. Native PAGE analysis of the formation of the triplexdependent tetrahedron at both pH 5.0 and 8.0. DNA sample compositions are indicated above the gel images and the DNA complex identity of each band is indicated at the sides of the gels. Note the dramatic mobility change for sample of L+M+S (1:3:3), as indicated by arrowheads.
Figure 3. Dynamic light scattering studies of the triplex-containing DNA tetrehedron. Figure 1. Reversible assembly/dissociation of a DNA tetrahedron in response to solution pH changes. (a) DNA three-point-star motif, consisting of one copy of L strand, three identical copies of M strands, and three identical copies of S strands, can associate with each other to form a DNA tetrahedron in acidic solution through DNA triplex formation of cytosine-containing sticky-ends (colored purple). A DNA tetrahedron consists of four copies of the three-point-star motifs and one of them is highlighted green. Solid red circles indicate the areas involving triplex-cohesion. The DNA tetrahedron will dissociate in neutral pH. (b) The detailed structure of one strut of the DNA tetrahedron. The red shaded area indicates the triplex-cohesion between two three-point-star motifs, which are related with each other by a 2-fold rotational axis that is perpendicular to the DNA plane and goes through the strut center. (c) DNA sequences of the strut center. Vertical lines and open circles indicate Watson−Crick and Hoogsteen base pairings, respectively. (d) Structure of one C+◦G-C triplet at pH 5.0.
Figure 4. Cycling operation of the smart DNA tetrahedron. DNA complex identities are indicated at the left. The flow of pH circling of the sample shows on the top of the gels and the numbers represent the sequential operation stages.
processing software developed by NIH) with very similar mobility as previously characterized tetrahedron marker. The formation of the triplex-containing DNA tetrahedron was further confirmed by dynamic light scattering (DLS). The tetrahedron sample appeared to have a hydrodynamic radius (Rh) of 10.2 ± 1.2 nm (Figure 3). The value was closely matched with the hydrodynamic radius (10.3 nm) of the previous characterized tetrahedron marker26a and was close to the radius (10.9 nm) of the circumscribed sphere of the DNA tetrahedral model, assuming 0.33 nm/basepair and 2.0 nm for the pitch and diameter of the DNA duplex, respectively. The triplex-containing sticky ends allowed the smart DNA tetrahedron to reversibly form and dissociate upon solution pH value oscillation between 5.0 and 8.0 (Figure 4). We started the
cycling operation by assembly of the DNA complex at pH 8.0 and then used 1.0 M HCl or NaOH to adjust the solution pH value. Before each round of pH adjustment, one aliquot of the DNA sample was withdrawn for analysis by PAGE at both pH 5.0 and pH 8.0. As the circling went, the DNA samples clearly changed its electrophoretic mobility at those pH values, strongly indicating that the DNA complex changed structures between individual motif (at pH 8.0) and tetrahedron (at pH 5.0). The assembly/dissociation could go at least four circles. We noticed that, with the progressive circling, the DNA band intensities decreased, presumably due to certain extent of DNA B
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aggregation during cycling (such large DNA aggregates accumulated in the well and could not penetrate into the gel matrix). In the smart DNA tetrahedron, the intermotif association has two components: (1) a two-base-long Watson−Crick, B-DNA, sticky-end hybridization and (2) a six-base-long triplex. The two base-long, sticky-end association directs the local geometry (linear arrangement of the DNA duplexes and the twist angles of the duplexes) of the intermotif association. Such a short sticky-end association is not stable by themselves under the current experimental condition and would readily dissociate. However, it can be stabilized by the pH-triggered triplex formation. On the other hand, the triplex alone is not suited for the construction of tetrahedron because of the structural complexity of the duplex−triplex junction, which presents a difficulty for structural prediction and control. In fact, they formed random large aggregates. In the current design, it is the right combination that leads to the successful construction of the smart DNA tetrahedron. In summary, we have developed a strategy to reversibly assemble/disassemble DNA nanocages based on pH-sensitive DNA triplex. We believe that the current design strategy can be readily adapted for other DNA nanocages, such as octahedron and icosahedron. The reported method increases the versatility of the toolbox of DNA nanoconstruction. We envision that such smart DNA nanocages could potentially capture and release cargos on-demand.4,5 Research in this direction is actively pursued in our laboratory.
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ASSOCIATED CONTENT
* Supporting Information S
DNA sequences and experimental details. 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]. Author Contributions Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Office of Naval Research for supporting this research. DEDICATION We would like to dedicate this communication to the celebration of the marriage of Mr. Zhiyu Liu and Miss Wenshi Tan.
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REFERENCES
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