Article pubs.acs.org/JPCB
Tetrahelical Monomolecular Architecture of DNA: A New Building Block for Nanotechnology Besik Kankia* Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *
ABSTRACT: DNA nanotechnology typically relies on Watson−Crick base pairing as both a recognition and structural element. This limits structural versatility and introduces errors during self-assembly of DNA. Guanine (G) quartet motifs show promise as an alternative to DNA duplexes, but the synthesis of long, precisely defined molecules is a significant challenge. Here we demonstrate a continuous tetrahelical DNA architecture capable of programmed self-assembly. We report that the homopolymer consisting of (G3T)3G3 monomeric units has the capability to fold into a monomolecular DNA tetrahelix with unprecedented speed and stability. For instance, in the presence of 1 mM K+ ions the dimer, (G3T)2, folds readily and melts above 100 °C. These findings have the potential to revolutionize DNA nanotechnology by introducing fast and error-free self-assembly of long and extraordinarily stable molecules.
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INTRODUCTION The remarkable self-assembly properties of DNA make it an excellent nanoscale material. However, DNA nanotechnology relies primarily on DNA duplex alignment.1−6 Guanine (G) quartets or quadruplexes, four guanines held together by Hoogsteen hydrogen bonding, show promise as an alternative to DNA duplexes. They had been proposed earlier as a potential tool for nanoscale assembly.7−11 However, the formation of long, precisely defined quadruplexes is a significant challenge. Specifically, since the G-quartets are formed by guanines only, it is problematic to prevent slippage of the strands relative to each other similarly to a DNA duplex made of homopolymers. As a result the annealed product is a complex mixture of stacks of G-quartets of different lengths.9−11 To solve this problem, quadruplexes were combined with DNA duplexes.12−14 However, this approach produces only short segments of intermolecular quadruplexes. Recently, we discovered that the free energy of a intramolecular (G3T)3G3) quadruplex, abbreviated to G3T, can be used to drive endergonic reactions, such as the polymerase chain reaction (PCR) at constant temperature.15 To increase driving force of such reactions, we proposed to design energetically more favorable DNA quadruplexes. Here, we explore the design and use of DNA quadruplexes in driving nanoparticle assembly. Since G3T represents the most stable monomolecular DNA quadruplex,16 we decided to use it as a monomer unit in building more complex structures. The study revealed that G3T dimerization through the nucleotide linkers (e.g., G3T-T-G3T) did not have a significant effect on the melting behavior relative to the free monomers. However, dimers and higher degree polymers, in which G3T monomers are directly attached to © XXXX American Chemical Society
each other without a linker revealed unexpectedly high thermal stability. Additional optical and kinetic studies suggested formation of monomolecular uninterrupted DNA structure based on the G-quartet. On the basis of the present studies and structural properties of G3T quadruplex,16,17 we suggest a model of tetrahelical monomolecular architecture of DNA. The model represents the only architecture, besides the Watson− Crick duplex, capable of forming an uninterrupted polymer in a programmable manner. In addition, it is the first monomolecular architecture able to form an infinite polymer. The present finding will create many opportunities in DNA nanotechnology as a building material and a driving force for the reactions.
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MATERIALS AND METHODS The DNA oligonucleotides were obtained from Integrated DNA Technologies: G3T (5′GGGTGGGTGGGTGGG) or (5′G3TG3TG3TG3); G3T-T-G3T ( 5 ′ G 3 TG 3 TG 3 TG 3 TG 3 T G 3 T G 3 TG 3 ); G3T-TT -G 3T (5′G3TG3TG3TG3TTG3TG3TG3TG3); (G3T)2 ( 5 ′ G 3 TG 3 TG 3 TG 6 TG 3 TG 3 TG 3 ); (G3T) 3 ( 5 ′ G 3 TG 3 TG 3 TG 6 TG 3 TG 3 TG 6 TG 3 TG 3 TG 3 ); (G3T) 4 ( 5 ′ G 3 TG 3 TG 3 TG6TG3TG3TG6TG3T-G3TG6TG3TG3TG3). Sequences of G3T-A-G3T, G3T-C-G3T, G3T-T3-G3T, and G3T-T5-G3T are listed in Supporting Information, Figure S1. The concentration of the DNA oligonucleotides was determined by measuring UV absorption at 260 nm as described earlier.18 Unless otherwise noted, all measurements were performed in Received: April 2, 2014 Revised: May 16, 2014
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two-state transition, with the melting temperature, Tm, of 75 °C.16 Dimerization through the thymidine linkers did not have a significant effect on the melting behavior relative to the free monomers. Since the melts are conducted at the same strand concentration, the dimers melt the same as a 2-fold concentrated solution of G3T monomers (Figure 1a). In contrast, a construct in which G3T monomers are directly attached to each other without a linker, (G3T)2, reveals a strong stabilization effect. The stabilization is so strong that in the presence of 1 mM K+ ions, the structure starts unfolding only above 95 °C. Experiments performed in the presence of only 100 μM K+ ions, confirmed that direct dimerization of G3T has a dramatic effect on the melting profile; the (G3T)2 molecule melts as a single domain with increased cooperativity and the Tm is shifted 30 °C higher than G3T (Figure 1b). Next we tested a trimer and a quadromer, (G3T)3 and (G3T)4. In each case a further increase in Tm was observed (Figure 1b). Thus, direct dimerization or polymerization of G3T monomers results in a novel and unusually stable structure, while insertion of T or TT links between G3T units completely inhibits formation of the structure. Additional UV melting experiments revealed that the inhibition effect does not depend on the type of the linker nucleotide (A, T, or C) or the linker length (T−T5) (Supporting Information, Figure S1). This unusually high thermal stability of the G3T multimers makes thermal unfolding experiments unfeasible. Therefore, we switched to isothermal experiments to monitor quadruplex formation by adding K+ ions into the solutions of unstructured sequences. We selected Cs+ ion as a counterion for the unstructured molecules, since it does not favor formation of Gquartets.18 UV melting experiments in the presence of Cs+ ions revealed that all constructs are unfolded above 50 °C (Supporting Information, Figure S2). Stopped-Flow Kinetics. Quadruplex folding rates are a useful parameter to estimate molecularity of the structures. The monomolecular quadruplexes fold readily and the folding rate does not depend on strand concentration. In contrast, the process of folding of a multimolecular quadruplex is extremely slow and depends on strand concentration. For instance, the half-life of TG4T tetraplex formation at 10 μM concentration is >100 days, while at 1 mM concentration the tetraplex folds within seconds.20 In CD measurements, conducted at 2−8 μM concentrations, quadruplexes were folded within seconds, as evidenced by lack of change in the signal following the addition of K+ and immediate spectral measurements. This supports the monomolecular fold. Stopped-flow kinetic measurements of (G3T)2, (G3T)3, and (G3T)4 also demonstrated fast folding rates of ∼7 s−1 independent of the strand concentration (Figure 2 and Supporting Information, Figure S3). Thus, is no doubt that G3T multimers fold into monomolecular quadruplex structures. CD Spectroscopy. In the presence of K+ ions, the CD spectrum of the G3T monomer shows a positive signal at 260 nm and a negative signal at 240 nM (Figure 3a). This is typical for all-parallel G-quartets with exclusively anti glycosyl bonds 21,22 formed by chain-reversal single-nucleotide loops.16,23 Antiparallel quadruplexes demonstrate completely different CD spectra: positive bands with maxima at ∼245 nm and ∼295 nm and a negative peak at ∼265 nm.21,24,25 Thus, even a partial change in the G3T folding upon multimerization should induce visible effects in CD spectra. The overall shapes of the CD spectra for all the multimers are very similar to the
10 mM Tris-HCl, pH 8.7, with the ionic strength adjusted by addition of appropriate salts as indicated in the figure legends. CD spectra were obtained with a Jasco-815 spectropolarimeter at using 2−8 μM oligonucleotide solutions in 1 cm cells. UV melting experiments were recorded on a Varian UV−visible spectrophotometer (Cary 100 Bio) at 295 nm with a heating rate of 0.5 °C per min. The optical devices were equipped with thermoelectrically controlled cell holders. In a typical experiment, oligonucleotide samples were mixed and diluted into the desired buffers in optical cuvettes. The solutions were incubated at 95 °C for 2 min in the cell holder prior to ramping to the desired temperature. The stopped-flow kinetics was studied with a STA-20 rapid mixture. The melting curves allowed an estimate of melting temperature, Tm, the midpoint temperature of the unfolding process. Van’t Hoff enthalpies, ΔHvH, were also calculated using the following equations for monomolecular structures: ΔHvH = 4RTm2(δα/δT). R is the gas constant and δα/δT is the slope of the normalized optical absorbance or fluorescence versus temperature curve at the Tm.19
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RESULTS AND DISCUSSION UV Melting Experiments. Initially, we conjugated two G3T units through thymidine linkers, T and TT, and performed UV unfolding experiments in the presence of 1 mM K+ ions (Figure 1a). As expected, the optical density of the G3T monomer reveals a sigmoidal transition, characteristic of a
Figure 1. UV melting experiments of G3T multimers. (a) In the presence of 1 mM K+ ions, dimers connected to each other through thymidine linkers, G3T-T-G3T and G3T-TT-G3T, reveal identical profiles with slightly less stability than the G3T monomer. The construct with directly connected monomers, (G3T)2, does not unfold under these experimental conditions. (b) In the presence of 0.1 mM K+ ions (G3T)2 unfolds as one cooperative unit with Tm of 85 °C, demonstrating 30 °C stabilization upon dimerization. The trimer and quadromer, (G3T)3 and (G3T)4, do not unfold completely even in 0.1 mM K+. B
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Figure 2. Stopped-flow kinetics of (G3T)4 quadruplex formation at 0.5 μM (a) and 0.08 μM (b) concentrations. The oligonucleotide solutions in 10 mM Tris-HCl, pH 8.7 were mixed with 1 mM K+ ions at 50 °C. The rate constants of the quadruplex folding, k, were estimated from a single-exponential fit (solid line) of the experimental data.
Figure 4. Schematic diagrams of monomolecular G3T multimers. (a) Two G3T monomers conjugated through a nucleotide linker (green). Each G3T monomer is shown as three disks (G-quartets) formed by four G3 segments (red) connected to each other by chain-reversal Tloops (black). (b) Two G3T monomers directly attached to each other forming continuous stack of 6 G-quartet, which is interfered by the nucleotide linkers as shown in panel a. (c) Four G3T monomers directly attached to each other forming a continuous polymer.
profile of the G3T monomer, but the magnitude of the bands varies. As expected, molar ellipticity of G3T-T-G3T at 260 nm is approximately 2-fold greater than that of the G3T monomer (Figure 3b). However, the magnitudes for (G3T)2, (G3T)3, and (G3T)4 do not increase proportionally and reveal a 2.6-, 3.5-, and 4.5-fold increase, respectively. The extra CD signal is most likely due to the stacking interaction between G3T monomers, which is interfered by T-linker in G3T-T-G3T (see Figure 4a,b). Since the CD signal of DNA quadruplexes is due to the base stacking interactions between G-quartets, one can estimate molar ellipticity per stacking interaction from the values of free G3T monomer, 0.248 × 106 deg m−1 M−1 at 260
nm. In the case of extra stacking interaction between G3T monomers in (G3T)2 one would expect 0.248 × 5 = 1.24 × 106 deg m−1 M−1, which is very close to the experimental value. We would like to emphasize that all constructs demonstrate the same CD profile, which indicates that the stacking interaction between and within G3T monomers are very similar to each other. Similarity of the stacking interactions between and within G3T monomers is further supported by the thermal unfolding experiments (Figure 1). Specifically, in the case of weak interaction between G3T monomers one would predict two
Figure 3. CD spectra of G3T multimers at 50 °C. (a) G3T monomer reveals a typical profile for an all-parallel quadruplex with chain-reversal loops. Thinner lines, here and in other panels, correspond to measurements in the presence of 10 mM Cs+ and correspond to unstructured single strands. (b) Both dimers demonstrate qualitatively the same CD profiles. However, (G3T)2 (solid line) reveals a stronger magnitude than G3T-T-G3T (dashed line) due to extra stacking interactions between directly attached monomers. (c) (G3T)2 and (d) (G3T)4, respectively, demonstrate exactly the same profiles with increased CD magnitudes. C
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Figure 5. Suggested models of the monomolecular tetrahelical architecture: (a) Sequence of the (G3T)4. The Arabic and Roman numerals correspond to nucleotide and G3T monomer positions. Color code is the same for all panels and helps to identify helices. For instance, the red helix is made of 13−18, 35−37, and 54−56 guanines with two interruptions (between 18 and 35, and 37 and 54). Three-dimensional models of the structures visualized for τ(G-G) = 30° (b) and 36° (c) (see text). The tetrahelical structure is viewed as a cylinder formed by stacked G-quartets (disks). The colored spheres represent sugars of guanines involved in G-quartets. The black spheres represent sugars of unstructured thymidines forming the chain-reversal loops (thymines are omitted for clarity). The right panels correspond to the three-dimensional models in the unfolded state.
quadruplex. Figure 4 shows topology of the proposed model. It represents vertical stacks of all-parallel G3T quadruplexes or monomers. Each monomer is formed by G3 segments connected to each other by chain-reversal T-loops in a propeller-like arrangement.17 G3T monomers are linked to each other by direct attachments of the terminal G3 segments forming uninterrupted G6, which is sheared by adjacent monomers (see Figure 4). For instance, the second G3T monomer shears front-left G6 with the first monomer and backleft G6 with the third monomer (Figure 4c). As discussed above, uninterrupted G6 is essential for formation of the tetrahelical polymer; incorporation of other nucleotides in the middle of G6 inhibits stacking between the monomers (Figure 4a,b). Kotlyar et al. reported earlier preparation of unusually stable monomolecular G-wires from poly(dG), which they concluded
separate transitions; initial destacking of G3T monomers at 30° it forms a right-handed helix; and when τ(G-G) < 30° it forms a left-handed helix. The suggested model has several distinct characters. While, the sugar−phosphate chain follows a zigzag pattern, the architecture is still helical. Each helix is formed by a discontinued sugar−phosphate backbone. For instance, the blue helix is made of four separated G3 segments, while the green helix is made of two G3 and one G6 segments (Figure
formed a long antiparallel quadruplex with three lateral loops.9 However, the reported CD spectrum is characteristic of a parallel quadruplex. Interestingly, quadruplex formation was accompanied by a 5-fold reduction in the length of initial poly(dG) instead of the expected 4-fold reduction.9 The discrepancy is perfectly explained by a tetrahelical model in which exactly 1/5 of the nucleotides (3 out of 15) are located in chain-reversal loops. Since G3T quadruplex tolerates nucleotide exchange in loop positions,26,27 it is quit possible that in their experiments poly(dG) adopted the tetrahelical structure suggested here. Before considering properties of the three-dimensional model of the tetrahelical DNA (Figure 5), the helical twist within and between G3T monomers should be discussed. There are two kinds of helical twists in G3T quadruplex: (i) helical twist between neighboring G-quartets, τ(G-G), such as G1-G2, G2-G3, G5-G6, etc.; and (ii) between top and bottom G-quartets upon chain-reversal T-looping, τ(G-T-G), such as G3-T-G5, etc. (see Figure 5b). While τ(G-G) is strictly determined by π−π stacking interactions between the guanines, τ(G-T-G) does not involve any direct interactions and is limited only by T-loop length. Since the first guanines of each G3 segments (e.g., G1, G5, G9, and G13) are the binding partners within the same G-quartet, the overall helical twist between them should be equal to 90° (e.g., τ(G1-G5) = τ(G1G2) + τ(G2-G3) + τ(G3-T-G5) = 90°) (see Figure 5b). The same is true for τ(G5-G9) and τ(G9-G13). However, the helical twist between G13 and G16 (first guanine of the next monomer), τ(G13-G16), equal to 3 × τ(G-G) and could be different from 90°. In summary, a helical turn between first E
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5a). All four grooves between the sugar−phosphate backbones are interrupted by T-loops forming one longer groove along the G6 segments and two shorter ones along G3 segments. This creates at least two categories of surface. First, mostly a negatively charged surface around the G6 segments, which occupies slightly less than half of the total surface. Second, the remainder of the surface with extra hydrophobicity due to exposed thymines. In the first model (Figure 5b) with τ(G-G) = 30°, which is missing helicity in terms of G3T units, the boundaries between the surfaces are straight lines creating halfcylinders with separated charged and hydrophobic surfaces. However, when τ(G-G) ≠ 30°, the boundaries rotate and follow helical pattern of G3T monomers (Figure 5c).
The free energy of DNA quadruplexes can be used to drive unfavorable (endergonic) reactions of nucleic acids (e.g., isothermal PCR).15 The key aspect of these reactions is that a (G3T)3G3 sequence forms a monomolecular quadruplex with significantly more favorable thermodynamics than the corresponding DNA duplex. Initially G3T or its modified version is incorporated within a DNA duplex. Upon interaction with an initiator (i.e., DNA polymerase, restriction endonuclease, or a binding partner) G3T self-dissociates from the complementary strand and folds into a quadruplex.15,31,32 One of the main reasons of the present study was to increase the driving force of such reactions by designing energetically more favorable DNA quadruplexes. The resulting 30 °C stabilization effect upon a simple dimerization of G3T monomers exceeds our expectations and paves the way to broader utilization of the quadruplexes as a fuel for various endergonic reactions.
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CONCLUSION This work suggests formation of a novel monomolecular architecture of DNA based on G-quartets as a structural element. This is the only architecture, besides the Watson− Crick duplex, capable of forming an uninterrupted polymer in a programmable manner. In addition, it is the first monomolecular architecture able to form an infinite polymer. We would like to emphasize that the monomeric unit, G3T, of the architecture is the 15-nt long sequence (G3T)3G3, not G3T. Therefore, the polymer, ((G3T)3G3)N, is a modified version of (G3T)N with every fourth thymidine deleted. The deletions create G6 segments, which are essential for the structure and can be used to program a desired construct. For instance, the structure of the sequence, (TG3TG3TG6)4 (another way to represent the novel polymer) can be precisely predicted as 3 units of G3T with unstructured tales at the both ends: 5′-TG3TG3TG3(G3T)3G3 (Supporting Information, Figure S4a). A simple attachment of G3 at the 5′-end would result in 4 units of G3T with only 3′-tale: 5′-(G3T)4G3 (Figure S4b). Several G → C substitutions will inhibit G3T quadruplex formation and the unfolded segment can be used for hybridization with another DNA strand (Figure S4c). Thus, the monomolecular tetraplex has strictly determined selfassembly properties similar to the DNA duplex. Moreover, the structure has several obvious advantages over DNA duplexes, which currently represents the main building material of DNA nanotechnology. The advantages include (i) monomolecularity of self-assembly, which accelerates the process, eliminates errors characteristic of bimolecular duplex formation and allows very simple production of DNA nanostructures through enzymatic replication (Figure 6); (ii) the ability to be folded/unfolded with little change in ionic strength (i.e., adding 0.1 mM K+ into 50 mM Cs+), which allows quadruplex formation without affecting other structures (i.e., DNA duplex); (iii) the folded structure is a tenth of the size of the original single strand, which could be used to induce movements in nanomachinery;7,30 (iv) the external mononucleotide loops allow for a simple incorporation of reporter molecules15 or binding partners (i.e., biotin to create a junction); (v) simplified multiarm junctions, which determines diversity of the final nanostructures. For instance, to create a 6way junction, six DNA strands should hybridize simultaneously (see Supporting Information, Figure S5a). The junction requires very careful design to avoid nonspecific interactions and branch migration. The junction in tetrahelical DNA forms significantly simpler by hybridization of short segments of single stranded regions between already preformed quadruplexes (Figure S5b).
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ASSOCIATED CONTENT
S Supporting Information *
Five additional figures including UV melting curves of G3T dimers, UV melting curves of all multimers in the presence of CsCl, stopped-flow kinetics of quadruplex formation, DNA sequences, and corresponding schematic models, and models of junctions in double-helical and tetrahelical DNA nanotechnology. 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]. Tel.: 614-688-8799. Fax: 614-6885402. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The author thanks Karin Musier-Forsyth for discussions and critical reading of the manuscript. This work was funded by a grant from the Bill & Melinda Gates Foundation through the Grand Challenges in Global Health initiative.
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REFERENCES
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