Letter pubs.acs.org/NanoLett
G‑Quadruplexes Light up Localized DNA Circuits Oscar Mendoza,*,†,‡ Jean-Louis Mergny,†,§ Jean-Pierre Aimé,†,‡ and Juan Elezgaray*,†,‡ †
Université de Bordeaux, 33600 Bordeaux, France CBMN, CNRS UMR-5248, F-33600 Pessac, France § Inserm, U1212, CNRS, ARNA laboratory, IECB, F-33600 Pessac, France ‡
S Supporting Information *
ABSTRACT: DNA circuits tethered to nanoplatforms can perform cascade reactions for signal amplification. One DNA single strand activates a stranddisplacement cascade generating numerous outputs, and therefore amplifying the signal. These localized circuits present, however, an important limitation: the spontaneous activation of the cascade reaction. Current methods to stabilize these circuits employ combination of protective DNA strands, which need to be removed to activate the device. This protection−deprotection process generates an important amount of unwanted side reactions. This is indeed an important limitation for the large potential application of these amplification circuits. In the present work, G-quadruplex DNA structures were used to stabilize localized DNA circuits. This new protocol generates nanoplatforms that no longer requires protective−deprotective systems and is therefore completely neutral to the sample. In addition, cations such as Pb2+ or Ca2+ can be also employed to activate the device enlarging the potential applications from biosensors devices to metal detector sensors. KEYWORDS: DNA strand displacement, G-quadruplex, localized amplification circuits (LAC), DNA origami
D
detection of short DNA sequences. In an amplification circuit, besides the three strands of a generic DSD reaction, an additional fuel strand (F) is introduced. As mentioned before, the strand I displaces the output O leading to the formation of I-G duplex. Then fuel strand F displaces the input I leading to F-G duplex and by doing so the input I strand becomes active to act into another G-O substrate (eq 1).
NA is a powerful and versatile material for nanoscale selfassembly. Because of its simplicity, the highly specific Watson−Crick hydrogen bonding allows convenient programming of DNA strands for the construction of nanostructures.1 Another attractive feature of DNA is the relative mechanical rigidity of double helices that can be employed as a semirigid spacer between structures connected at both ends of the double helix.2 However, the potential applications of DNA selfassembly are not limited to building blocks for nanomaterials construction. Double-stranded helical structures can be also applied for the construction of molecular programming circuits3−6 In these circuits, a correct design of DNA sequences can be used to store state and monitor it via strand displacement reactions.7 In a DNA strand-displacement reaction (DSD) a strand called “output” (O), which is hybridized with a complementary sequence (strand called “gate”, G) is displaced by a strand called “input” (I) leading to the formation of a gate-input duplex (I-G).7 DNA circuits show promise for applications in biosensors,8,9 nanorobots,10 or DNA computing.11−13 However, their implementation as a set of bulk reactions faces several difficulties, which include slow (diffusion limited) reaction times and unwanted cross-talk reactions. Constraining distances between different components of a circuit could solve these two difficulties. Following this idea, previous reports showed how DSD circuits can be localized on individual DNA-based nanoplatforms such as origamis14,15 for the nanodesign of localized amplification circuits (LAC).16 In an amplification circuit, one input strand triggers a cascade of reactions generating several output strands. These amplification circuits are clearly important for the development of biological sensors for the © XXXX American Chemical Society
I + GO + F → IG + F + O IG + F + O → I + GF + O
(1)
where I is the input strand, F is the fuel strand, G is the gate strand, and GO, IG, and GF are duplex DNA motifs formed by these complementary sequences. Amplification circuits attached to nanoplatforms present clear advantages to similar DSD reaction carried out in bulk solutions (thus without I, GO, and F connected to an individual platform): (i) reaction time of DSD reactions in solution is a factor of strand concentration (which at nanomolar scale can lead to long reaction times), while the response time is much faster between tethered strands; (ii) therefore, for a given time scale the required concentration of DNA substrate is reduced with respect to bulk reactions; (iii) DSD reactions on LAC platforms are predetermined not only by the DNA sequence but also by their location on the nanostructure; and (iv) side reactions are less likely to take place. Two types of circuits involving coupled DSD reactions have been reported so far. Walkers on geometrically prescribed Received: October 27, 2015 Revised: December 22, 2015
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DOI: 10.1021/acs.nanolett.5b04354 Nano Lett. XXXX, XXX, XXX−XXX
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Here we report how G-quadruplex DNA structures can be used to stabilize tethered DNA circuits without requiring the presence of strand protectors. G-quadruplexes are noncanonical nucleic acid structures formed by guanine-rich DNA or RNA sequences; they rely on the stacking of two or more G-quartets in which four guanines establish a cyclic array of hydrogen bonds (Figure 2). Cations such as Na+, K+, or NH4+ are located in the central channel of the structure and stabilize the assembly. Playing with the strand sequence and cation concentration (especially K+ or NH4+) allows the modulation of G-quadruplex thermal stability. Indeed, the concentration of the corresponding cation can be employed to switch from an unfolded single-stranded sequence to a G4 structure in a reversible manner with potential applications in the sensors field.18,19 Following this idea, stable single-stranded G-quadruplex sequences were integrated into the input and fuel strands (Figure 1B) in a manner that strand-protections were no longer needed. In order to activate these G4 substrates, a reduction of the cation concentration (K+ in this study) was sufficient. The LAC nanoconstruction comprised DNA strands (I, F, and G-O) connected to a DNA origami platform. The LAC construction considered in this research was formed by one input I and four F and G-O substrates located nearby (Figure 3A), which were connected to the origami by elongation of one strand end (see Supporting Information). In an initial state, gate and output strands are fully hybridized forming the G-O substrate (Figure 3A), while single-stranded I and F are folded into a G-quadruplex motif. Upon activation of the origami platform (Figure 3B), I 3′end can recognize the 5′end of G and displace the output strand. As O strand is not connected to the LAC platform, it is released to the solution. At this stage, the 5′end of F strand can recognize the 3′end of G and therefore release the input I. This can then act on another G-O+F system and release the four O strands from the LAC platform after four cycles (Figure 3C).
circuits have their driving force provided by an external input, such as enzymes.17 These systems remain inactive as long as the initial input and the enzymes are absent. The speed of reaction in walker systems is limited by the enzymatic turnover and the enzyme concentration. On the opposite, when the driving force (here, the fuel strand) is part of the circuit (as in LAC platforms, ref 16) the initial state of the circuit is intrinsically metastable (because the “effective” concentration of fuel is extremely high as LAC can be considered as a “pseudo” intramolecular assembly). This ensures a fast response but also requires the protection of the initial state. As in the walker system, two signals are needed to start the LAC platform, an input strand and deprotecting strands (Figure 1A). As a matter
Figure 1. (A) Gate substrates participating in a classical DSD reaction: input (red) and fuel (blue) are hybridized by protectors strands (black). (B) G-quadruplex protection: input (red) and fuel (blue) are folded in a G-quadruplex motif. Reducing the concentration of K+ destabilizes the G4 structure activating the circuit.
of fact, connecting DSD induces a high degree of similarity between sequences. From this point of view, ensuring that deprotecting sequences only remove the protections and do not trigger other parts of the circuit adds additional difficulties in the design and limits the size of these circuits.
Figure 2. (A) Representation of a G-tetrad; four guanine nucleotides are associated together via Hoogsteen hydrogen bonds. (B) Schematic representation of an intramolecular G-quadruplex. B
DOI: 10.1021/acs.nanolett.5b04354 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 4. (A) Real-time emission enhancement of LAC platform after activation: 1, 0.75, 0.5, 0.25, 0.15, and 0.1 μL of LAC nanodevice are added to 100 μL of buffer solution (final KCl concentration is therefore 200, 150, 100, 50, 30, and 20 μM respectively). (B) Normalized signal of the LAC platform after activation (t = 0) and addition of an important excess of input sequence (at t = 90 min for 0.25, and 0.5 μL of LAC platform, and t = 120 min for 1 μL).
rate of several minutes. In addition, the operating temperature for the LAC platform was optimized at 35 °C, as lower temperature produced even longer reaction times. Expectedly, the fluorescence enhancement was larger when more FAMlabeled origami was added to the solution. The kinetic response was also dependent on the origami volume added, which is due to the KCl present in the mixture and not to the origami concentration in itself. This localized DSD circuit can be considered as an intramolecular assembly, thus the kinetic reaction is independent of the substrate concentration. The reaction pathway of this tethered circuit is remarkably complex. The overall process involves several reactions: unfolding of quadruplex structures (I and F), interaction of I and F with G-toeholds, and several displacement reactions with both polarities (5′ to 3′ and 3′ to 5′). In addition, there are some leak reactions that potentially can affect the overall reaction (see below). Therefore, a deep and rational study of the kinetic behavior of these devices is difficult to accomplish. However, the global reaction could be simplified in two main reaction steps: first, the abrupt decrease of [K+] induces G4 unfolding; and second, strands interact through toehold mediating strand displacement reactions. Whereas the former process is strongly dependent on [K+], the later can be assumed to be approximately [K+] independent. In ref 16, we already considered kinetic modeling of the coupled DSD reaction that successively release the output strand to bulk. Two conclusions could be derived from this modeling: the high (mM) effective concentrations due to the pinning of gates to origamis induce very fast toehold mediated kinetics, which were estimated to be faster than 1 s−1 rates. It can be then considered that in this G4protected device the limiting factor is the unfolding process of the quadruplex structures located in I and F, while the followed DSD reaction is much faster.
Figure 3. (A) LAC nanoconstruction considered in this research formed by one input (red) and four fuel (blue) substrates folded into G-quadruplex motives surrounding four G-O duplexes (yellow); (B) active form after unfolding of the G4 structures; (C) final state after the four output strands are released.
In our design, the DSD circuit reaction was monitored in real time by simply labeling the O and G strands with a fluorophore and quencher, respectively. The output strand 5′end was labeled with fluorescein dye FAM while the 3′end of the gate strand was labeled with the quencher dabcyl. Therefore, in the initial stage of the LAC platform, the four output strands are hybridized with the four gate strands quenching the fluorescence of the labeled output. When the DSD circuit is triggered, O strands are progressively released to the bulk solution, recovering the fluorescence of the organic dye. This allowed monitoring the DSD reaction in real-time. Assembled LAC circuits (see Supporting Information for assembling protocol) were stored at 20 mM KCl buffered solution as an “inactive” circuit. The activation was done by direct addition of LAC solution to a buffer solution containing no K+ cations. This reduced significantly the K+ concentration of the final mixture (from 20 mM down to 20−200 μM), unfolding (or reducing considerably the stability of) the Gquadruplexes present in I and F substrates (A → B in Figure 3). This allowed the recognition between I and G-O substrates triggering the DSD reaction. Figure 4A shows the LAC device response when 0.1, 0.15, 0.25, 0.5, 0.75, and 1 μL of LAC stock solution were diluted in 100 μL buffer containing no KCl. Although K+ cation dilution was pronounced (final K+ concentration of 20, 30, 50, 100, 150, and 200 μM, respectively) the response time was found in the C
DOI: 10.1021/acs.nanolett.5b04354 Nano Lett. XXXX, XXX, XXX−XXX
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Nano Letters Individual fitting models were applied to the reactions shown in Figure 4A (Supporting Information Figure S1) and reaction rate constants (k) were calculated considering this limiting step as an intramolecular process (Table 1). Unsurprisingly, the calculated values (Table 1) shows a clear trend: the higher the [K+] value, the lower the speed of the reaction. Table 1. Calculated Reaction Rate Constants of the LAC Platform Response at Different KCl Concentrations and 35°Ca [KCl] (mM) 20 30 50 100 150 200 a
reaction rate constant k (s−1) (2.08 (1.29 (8.85 (4.02 (2.73 (2.18
± ± ± ± ± ±
0.28) 0.33) 0.19) 0.28) 0.03) 0.11)
× × × × × ×
10−5 10−5 10−6 10−6 10−6 10−6
Figure 5. Normalized signal of the real-time emission enhancement of LAC platform containing the following: all the F, G-O, and I substrates (blue); G-O and F strands (green); and I and G-O substrates. DSD circuit initiated after addition of 1 μL origami stock to 100 μL buffer (thus reducing the KCl concentration to 200 μM) of the LAC platform after activation (t = 0) and addition of an important excess of input sequence (at t = 120 min).
Errors calculated from the average of two independent repetitions.
It can be then concluded that the switch single-strand G4 can be employed effectively to trigger a response of a LAC device without requiring strand protections and activator. The use of this noncanonical DNA structure to obtain stable LAC devices is an important novelty for the future design of DNA circuits tethered to platforms: as these structures will no longer require strand-protectors, there is no limitation to the number of DNA substrates that could be tethered to the platform. In addition, although in the present study K+ was used to trigger the LAC platform, several metal cations are able to stabilize a Gquadruplex (Rb+, Cs+, Sr2+, Ba2+, Ca2+, Pb2+, and so forth), which enlarge the potential applications of these LAC nanodevices to inputs other than nucleic acids.
To quantify the efficiency of the device, a second step was introduced in this DSD reaction. Once the DSD reaction was completed and the emission signal reached a stable plateau, the single-stranded I sequence was introduced to the mixture in a considerable excess (∼200 fold excess). This important excess of input sequence can displace and release any O strand still hybridized to a G substrate. The introduction of this second step allowed the quantification of the efficiency of this amplification circuit, finding a total yield of about 50%. (Figure 4B shows an example of this two-step DSD reaction). The response of the same DNA circuit not connected to an origami platform (thus I, F, and G-O substrates in bulk solution) was found to be considerably slower as no significant fluorescence output was observed after 1 h (Supporting Information Figure S2). As mentioned above, the DSD circuit comprises a series of DSD reactions involving input and fuel substrates. To verify the cooperative participation of F and I substrates two controls were carried out, an origami platform containing only the four G-O and F substrates tethered to the device (i.e., I strand not included in the assembly) and an origami platform containing only I and G-O substrates connected (i.e., fuel substrates not attached). As it can be observed in Figure 5, in the absence of fuel strands the input substrate can nevertheless release one output strand (Figure 5, red). While in the absence of the input system, the response time is extremely slow, and G-O gates require a long time to be displaced by the four F (Figure 5, green). This corresponds to a blunt end DSD reaction in which fuel has no available single-stranded segment to start the displacement. Therefore, the combination of the three substrates, I+GO+F, is at the origin of the amplification signal and thus the presence of both I and F is required to obtain a total response of the nanosensor. In the LAC device considered in this study, all the interactions between strands are toehold mediated. Depending on the GC content, the kinetic constants that characterize strand displacement are in the order of 105 M−1 s−1.20 Accordingly, the expected time scale of interactions between strands belonging to different origamis (assuming the total origami concentration in the reaction well is below 500 pM) is longer than 105 s. Therefore, interorigami interactions can effectively influence the long-time kinetic trend, which is not considered in this study.
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METHODS Oligonucleotides. DNA oligonucleotides used in this work were purchased from Sigma-Aldrich (origami staples, FAMlabeled output strand, input strand, and fuel strand) or Eurogentec (Dabcyl labeled gate strand). Origami staples (desalted quality) were used without further purification, while gate strands (input, output, fuel, and gate) were HPLC purified. All DNA oligonucleotides were stored in water at 100 μM concentration and at −20 °C. Concentrations were determined from the absorbance at 260 nm (Nanodrop Thermo Scientific) using the extinctions coefficients provided by the oligonucleotide manufacturer. M13mp18 was purchased from New England BioLabs. All experiments were done on Tris acetate-EDTA buffer (TAE, purchased as 10× stock from Sigma-Aldrich) supplemented with 12.5 mM magnesium acetate. When required, K+ was added from a 1 M stock solution of KCl. SD Reactions on the LAC Gate. These were carried out in 96-well plates (Greiner Bio-one 96-well black flat bottom) at 35 °C and the fluorescence monitored in a microplate reader (Tecan Infinite M1000 PRO, Lyon, France). Every well contained 100 μL of TAE buffer with no KCl in the mixture. After 10 min of temperature equilibration, previously prepared LAC platforms (∼100 nM) were added to every well, the 96well plate was stirred for 10 s, and the fluorescence emission was recorded every 5 s (the excitation wavelength was set at 492 nm and the emission wavelength at 520 nm). Once the maximum emission was reached and the signal was stable (60− 90 min), 2 μL (10 μM) of an input strand was added to every D
DOI: 10.1021/acs.nanolett.5b04354 Nano Lett. XXXX, XXX, XXX−XXX
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(13) Seelig, G.; Soloveichik, D.; Zhang, D. Y.; Winfree, E. Science 2006, 314, 1585. (14) Rothemund, P. W. K. Nature 2006, 440, 297. (15) Ke, Y.; Lindsay, S.; Chang, Y.; Liu, Y.; Yan, H. Science 2008, 319, 180. (16) Mullor Ruiz, I.; Arbona, J.-M.; Lad, A.; Mendoza, O.; Aimé, J.P.; Elezgaray, J. Nanoscale 2015, 7, 12970. (17) Wickham, S. F. J.; Endo, M.; Katsuda, Y.; Hidaka, K.; Bath, J.; Sugiyama, H.; Turberfield, A. J. Nat. Nanotechnol. 2011, 6, 166. (18) Lv, L.; Guo, Z.; Wang, J.; Wang, E. Curr. Pharm. Des. 2012, 18, 2076. (19) Yatsunyk, L. A.; Mendoza, O.; Mergny, J.-L. Acc. Chem. Res. 2014, 47, 1836. (20) Zhang, D. Y.; Winfree, E. J. Am. Chem. Soc. 2009, 131, 17303.
well, the plate was stirred for 10 s, and emission was monitored every 5 s. SD Reactions on Bulk Solution. These were carried out by preparing a mixture of 100 and 400 nM of input and fuel substrates, respectively (previously annealed in buffer containing 100 mM KCl), and 400 nM of previously annealed gateoutput duplex. The final concentration of KCl was adjusted to 20 mM. Therefore, the concentrations of I, F, and G-O substrates were similar to the concentration found in the LAC stock solution (but without the presence of the nanoplatform). Following previous procedure the corresponding volume of DNA substrates mixture was added to 100 μL of buffer solution containing no KCl. The well-plate was then stirred for 10 s, and the fluorescence emission was recorded every 5 s (the excitation wavelength was set at 492 nm and the emission wavelength at 520 nm). Once the maximum emission was reached and the signal was stable (60−90 min), 2 μL (10 μM) of an input strand was added to every well, the plate was stirred for 10 s, and emission was monitored every 5 s. (Supporting Information Figure S2)
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b04354. All experimental details and supplementary tables and figures cited above. (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: (O.M.)
[email protected]. *E-mail: (J.E.)
[email protected]. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
This work was supported by: Conseil régional d’Aquitaine, ANR Vibbnano [ANR-10-NANO-04−03],Oligoswitch [ANR12-IS07-001-01], CNRS and Inserm. Notes
The authors declare no competing financial interest.
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