G-quadruplexes light up localized DNA circuits.

Transcription

1 G-quadruplexes light up localized DNA circuits. Oscar Mendoza,*,, Jean-Louis Mergny,, Jean-Pierre Aimé, and Juan Elezgaray*,, Univ. Bordeaux, Bordeaux, France CBMN, CNRS UMR-5248, F Pessac, France INSERM, ARNA Laboratory, U869, IECB, F Pessac, France LAC device construction: As mentioned before, the aim of this research is to employ intramolecular G-quadruplex structures to inactivate the input and fuel strands and prevent spontaneous initiation of the LAC circuit. Therefore, I and F strands consist in G-rich sequences, while the gate strand is formed by the C-rich complementary sequence (Table S1). The aim is to generate G4 motifs involving the 5 and 3 end of F and I respectively, thus they are no longer accessible for any potential recognition of G strand. Two main factors regulate the stability of a G-quadruplex: the selected strand sequence and the cation concentration (K + in our study). G-quadruplex structures are formed by the stacking of several G-quartets connected by short loops. A characteristic highly stable quadruplex sequence contains at least three G-quartets and two short (1 nucleotide) loops, following the sequence pattern: 5 - GGGNGGGN (n) GGGNGGG-3. The input and fuel sequences were therefore selected following this design. As shown in Table S1, I, F and G share the same G-rich core while they contain different G- rich flanking sequences. This combination of several 3-guanine runs was found to form highly stable G4 motifs in I and F DNA strands even at low concentration of K + cation (see SI). The LAC nanoconstruction comprised the DNA seesaw (I, F and G-O) connected to a DNA origami platform. The origami platform consisted in a 2D DNA origami of approximate dimensions 90nm x 60nm. The original design of the origami was modified in a manner that the required origami staples were elongated by 3-nt poly(dt) (to confer some flexibility) and 15-nt connector-sequence in order to tether the DSD circuit (I, F and G-O substrates) by the formation of 15-bp connectors (foot). The LAC construction considered in this research was formed by one input I and four F and G-O systems located nearby. I, G and F strands were connected to the DNA origami by a 15-nt poly T linker, which confers some flexibility to the system, and a 15-nt termination, which connect the circuit to the origami (foot). I, F and G denotes the sequence moiety participating in the DSD reaction of input, fuel and gate respectively; thus without the linker and foot strand terminations (Figure S3). In the design considered in this study, I, O and F share a 16-nt sequence core which is fully complementary to gate G. In addition, I, O and F contain either 5 or 3 flanking sequences, which can recognize the strand G at the 3 or 5 termination respectively, and by doing so, activate the DSD cascade. First, gates (I, F and duplex G-O) were prepared independently. Input and fuel strands, which are folded into a G-quadruplex structure, were annealed at 10 µm concentrations in buffer supplemented with 100 mm KCl. The mixture was heated to 90 C for 5 min a cooling rapidly in ice. S1

2 Gate-output duplex substrate was also annealed at 10 µm in a TAE buffer containing no KCl by heating at 90 C and cooling down to RT in 2h. DNA origami was prepared by mixing 10 µl of M13mp18 with a 5-fold excess of origami staples in TAE buffer (with no KCl). The annealing process followed a two-ramp temperature profile: from 90 C to 60 C in 1 h and from 60 C to 20 C in 2 h. The folded origami was then stored at 4 C overnight. KCl was added to the solution to adjust its concentration to 100 mm. Then, gate substrates (input, gate-output and fuel previously annealed) were added in a 50-fold excess with respect to the M13mp18 phage and the mixture incubated at 4 C for 48h. After the incubation of the origami platform with the gate substrates (I, F and duplex G-O), TAE buffer was added to the mixture to adjust the concentration of KCl to 20 mm. Then, the LAC device solution was filtered over an Amicon Ultra 100K filter to obtain a pure LAC circuit of about 100 nm concentration (concentration determined as g/l using a Nanodrop Thermo Scientific and considering the MW of the platform to be 5x10 6 g/mol). When the described LAC nanoconstruction was prepared in the presence of 100 mm KCl, the input and fuel sequences were found to be completely inactive (thus folded into a G-quadruplex motif) and no DSD reaction was observed. The nanodevice was found to be stable at this K + concentration for weeks, thus this cation concentration can be considered as an optimal storage solution for the LAC platform. The activation of the LAC circuit was carried out by reducing significantly the KCl concentration. In a first step, KCl concentration contained in the buffer storage was reduced from a 100 mm KCl to a 20 mm. This K + concentration was also found to be considerably high to maintain inactive both I and F sequences for more than 3 days. Spectroscopic characterization of G-quadruplex structures Spectroscopic characterization of input and fuel sequences was carried out in order to confirm the correct folding of these sequences into a G-quadruplex structure. As it can be observed in Figure S4, the UV melting profile (monitored at 295 nm) shows a stable G4 structure for both I and G substrates when annealed in a buffer containing 20mM KCl. This confirms that the KCl concentration employed in the LAC stock solution is sufficient to maintain I and F completely folded into a stable G4 structure. However, these quadruplex motifs become severely affected at lower KCl concentration. In fact, at the KCl concentration employed in the reaction well (<200 μm) the G4 structure is no longer folded, as the Tm at this [K + ] is remarkably lower than the reaction temperature (35 C). These results are in agreement with the results observed in the origami-device: while at 20 mm KCl input and fuel are stable folded into a G4 motif (and therefore, inactive to interact with the G-O substrate), at low KCl the quadruplex are probably unfolded. Circular dichroism was employed to confirm the presence of a G4 motif in I and F substrates and to analyse their structural conformation. CD spectra were recorded for input and fuel sequence at the KCl concentration employed in the origami stock solution (thus 20 mm) and the final KCl concentration in the reaction well (20, 30, 50, 100, 150 and 200μM). As it can be observed in Figure S5, at 20mM KCl buffered solution both sequences show an intense positive band at 265 nm and a negative peak at about 240 nm (Figure S5, black line), which reveals the folding of a G-quadruplex DNA structure. However, as it was previously found by UV melting profiles, the G-quadruplexes are found to be disrupted at the reactional K + concentration (Figure S5, coloured lines). S2

3 In addition, I and F quadruplexes display a distinctive foot-print of a parallel G4 motif (Figure S5, black line). This is not unexpected as the selected sequences for I and F follow the pattern GGGNGGGNGGGNGGG, which usually folds into a parallel quadruplex. A CD characterization of I and F when tethered to the origami is unachievable. Duplex DNA is also optically active and exhibits characteristic intense bands in CD spectroscopy. This makes very difficult to study a single G-quadruplex structure when surrounded by a duplex motif. The nano-platform considered in this research contains more than 7000 W-C bp, while only five G-quadruplexes, making impossible to study by CD the quadruplex structures when tethered to the platform. Nevertheless, it is possible to calculate approximatively the CD spectra of a G-quadruplex adjacent to a short duplex DNA. This can be obtained by recording the spectrum of the G4-duplex substrate and subtracting the contribution of the duplex moiety (which is obtained independently). However, this approach is only feasible when the contribution of the duplex moiety to the CD signal is of the same order of magnitude than the G-quadruplex. 1 4 This is not the case in the origami platform as the duplex contribution is extraordinarily higher than the five G4 motifs tethered. However, the correct folding of I and F G4 structures connected to foot sequences could be studied applying this approach. For this purpose, input and fuel substrates were hybridized with their corresponding foot staples (but without being tethered to the origami platform). This leads to a G- quadruplex structure connected to a 15bp duplex foot (I-foot and F-foot respectively). CD spectra of I-foot and F-foot as well as foot duplex DNA substrate were recorded at the KCl concentration employed in the stock solution, thus 20 mm (Figure S6). As it can be observed, input and fuel connected to duplex foot (I-foot and F-foot) shows altered spectra when compared to I an F substrates (Figure S5 and S6). However, when duplex contribution is subtracted (Figure S6, black line) the final CD spectra recovers the original G4 pattern (Figure S6, dash lines). This confirms that G-quadruplexes remain stable when attached to the duplex DNA foot. METHODS Circular dichroism (CD) spectroscopy: Circular dichroism spectra were recorded on a Jasco J-815 equipped with a Peltier temperature control accessory (JASCO Co., Ltd., Hachioji, Japan). Each spectrum was obtained by averaging three scans at a speed of 100 nm/min. A background CD spectrum of corresponding buffer solution was subtracted from the average scan for each sample. UV-melting experiments. Melting experiments of input and fuel strands (comprising the linker an foot elongations) were examined by measuring the changes in absorbance at 295 nm as a function of the temperature, using a SAFAS UVmc2 double-beam spectrophotometer (Monte Carlo, Monaco) equipped with a high-performance Peltier temperature controller. (Figure S3) After heating at 90 C a sample containing 2µM of DNA substrate in 10 mm cacodylate buffer containing 50 nm, 100 nm, 200 nm and 20 mm KCl respectively at ph 7.2. The absorbance was monitored at 240, 260, 273, 295 and 335 nm on a cycle composed of a cooling down to 0.5 C at a rate of 0.7 C min 1. REFERENCES: (1) Mendoza, O.; Porrini, M.; Salgado, G. F.; Gabelica, V.; Mergny, J.-L. Chem. - A Eur. J. 2015, 21, S3

4 6732. (2) Zhou, J.; Bourdoncle, A.; Rosu, F.; Gabelica, V.; Mergny, J. L. Angew. Chemie - Int. Ed. 2012, 51, (3) Ying, L.; Green, J. J.; Li, H.; Klenerman, D.; Balasubramanian, S. Proc. Natl. Acad. Sci. 2003, 100, (4) Bagheri, Z.; Ranjbar, B.; Latifi, H.; Zibaii, M. I.; Moghadam, T. T.; Azizi, A. Int. J. Biol. Macromol. 2015, 72, 806. S4

5 Emission (a.u.) Table 1. Table 1. Oligonucleotide sequences used for the formation of the I, G-O and F system. System Output (O) Input (I) Fuel (F) Gate (G) Sequence FAM-5 -CGGGCGGGTAGGGTGGGTAGG-3 5 -GGGTAGGGTGGGTAGGGTGGG-TTTTT-(foot)-3 5 -(foot)-ttt-cgggcgggtagggtgggtaggg 5 -(foot)-ttttt-cccaccctacccaccctacccgcccg-3 -Dabcyl Figure S time (min) Figure S2. Raw data (black spots) and fitting model (red line) of the emission enhancement of LAC platform after activation (see Figure 4A in the main text) Figure S2. Figure S2. Normalized emission signal of the DSD reaction performed in bulk (thus I, G-O and F are not connected to an origami platform) after dilution of 0.25, 0.5 and 1 μl of substrates mixture to a 100 μl buffer with no KCl (t=0) and after addition of an important excess of input sequence (t= 60 min). S5

6 Figure S3. Figure S3. Schematic representation of DNA strand gate (in a 3 to 5 polarity) and the complementary output, input and fuel DNA sequences (in a 5 to 3 polarity).. Figure S4. Figure S4. Melting profiles (normalized absorbance at 295 nm) obtained for input I (A) and fuel F (B) substrates (2 µl) in 10 mm cacodylate buffer containing 50 nm (blue), 100 nm (red), 200 nm (green) and 20 mm KCl (orange) respectively at ph 7.2. Figure S5. Figure S5. CD spectra at 35 C of input I (A) and fuel F (B) in 10 mm cacodylate buffer (ph 7.2) containing either 20 mm KCl (black line) or the KCl concentration employed in the reaction well (20 to 200 µm) (coloured lines). S6

7 Figure S6. Figure S6. CD spectra at 35 C of I-foot substrate (A, red line) and F-foot substrate (B, blue line) in 10 mm cacodylate buffer (ph 7.2) containing 20 mm KCl (black line). The CD spectrum of a duplex DNA containing 15bp (foot) recorded under identical conditions (concentration, buffer and temperature) is shown in black. The dash line in figure A and B shows the subtraction of the CD spectrum of the foot duplex from the CD spectrum of the I-foot and F-foot respectively. S7