Reversible DNA i-motif to hairpin switching induced by copper(ii) cations

i-Motif forming DNA sequences have previously been used for many different nanotechnological applications, but all have used changes in pH to fold the DNA. Here it is shown that Cu(ii) cations can be used to re-fold i-motifs into hairpin structures, without changing the pH.


GENERAL EXPERIMENTAL
All the oligonucleotides (ODNs) and their fluorescent conjugates were purchased from Eurogentec and were HPLC purified. Solid DNA samples were initially dissolved as a stock solution in MilliQ water (100 μM for labelled and 1 mM for un-labelled ODNs); further dilutions were carried out in the respective sodium cacodylate buffer. Annealed samples were thermally annealed in a heat block at 95°C for 5 minutes and cooled slowly to room temperature overnight. Non-annealed samples had the DNA diluted into the respective buffer and were used immediately.

FRET MELTING EXPERIMENTS
The ability of cations to affect the stability of i-motif DNA was assessed using a fluorescence resonance energy transfer (FRET) DNA melting based assay. The labelled oligonucleotide hTeloC FRET (5'-FAM-d[TAA-CCC-TAA-CCC-TAA-CCC-TAA-CCC]-TAMRA-3'; donor fluorophore FAM is 6-carboxyfluorescein; acceptor fluorophore TAMRA is 6-carboxytetramethylrhodamine) was prepared as a 400 nM solution in buffer containing 10 mM sodium cacodylate (pH 7.4) with 5 mM NaCl and then thermally annealed. CuCl 2 was dissolved in purified water. Strip-tubes (QIAgen) were prepared by aliquoting 10 μL of the annealed DNA, followed by 10 μL of the cation solutions. Fluorescence melting curves were determined in a QIAgen Rotor-Gene Q-series PCR machine, using a total reaction volume of 20 μL. Measurements were made with excitation at 483 nm and detection at 533 nm. Final analysis of the data was carried out using QIAgen Rotor-Gene Q-series software and Origin.

CIRCULAR DICHROISM EXPERIMENTS
Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter using a 1 mm path length quartz cuvette. Human telomeric i-motif (hTeloC, 5'-d[TAA-CCC-TAA-CCC-TAA-CCC-TAA-CCC]-3') was diluted in a buffer containing sodium cacodylate (10 mM or 50 mM and pH 7.4 or 5.5 as detailed) to achieve a total volume of 200 µL. The scans were performed at 20°C over a wavelength range of 200-320 nm with a scanning speed of 200 nm/min, a response time of 1 s, 0.5 nm pitch and 2 nm bandwidth. A blank sample containing only buffer was treated in the same manner and subtracted from the collected data. Solutions of CuCl 2 and EDTA were added in small aliquots to the desired equivalent proportions using a pipette. The CD spectra represent an average of three scans and are zero corrected at 320 nm. Kinetics experiments were performed using the time course management function in the Jasco software using single measurements every 0.5 s at 288 nm. Each experiment was performed in triplicate. Final analysis and processing of the data was carried out using Origin.

UV EXPERIMENTS
UV spectroscopy experiments were performed on a Agilent Technologies Cary 60 UV-Vis spectrometer equipped with a Quantum Northwest TC1 thermal peltier controller and recorded using a low volume quartz cuvette. The thermal melting curves were obtained by monitoring the absorbance at 295 nm. Samples (200 µL) were prepared then transferred to a masked quartz cuvette (1 cm path length), covered with a layer of silicone oil and stoppered to reduce evaporation of the sample. Samples were held at 4°C for 5 minutes then heated to 95°C three times at a rate of 0.5°C/min, each with a 5 minute hold at 4°C and S4 95°C and data was recorded every 1°C during both melting and annealing. Each point was the average of three scans. Melting temperatures (T m ) were determined using the first derivative method. from the unfolded spectrum (in the absence of CuCl 2 ) and normalised so the maximum change in absorption was set to +1 as previously described. 4

1 H NMR EXPERIMENTS
1 H NMR experiments were performed using a Bruker Avance III 800 MHz spectrometer equipped with an HCN inverse triple resonance z-gradient probe. Aqueous solutions were S5 prepared with the addition of 5% D 2 O to enable field/frequency lock. Solvent suppression of the water resonance was achieved using a 1D Watergate sequence employing a symmetrical 3-τ-9-τ-19 pulse train inversion element. The solvent resonance, which was minimized, was set on-resonance at the transmitter offset and the interpulse delay time (τ) was adjusted to achieve an excitation maximum in the imino proton region of interest. The hTeloC oligonucleotide sequence was diluted to a concentration of 10 µM in pH 5.5 50 mM sodium cacodylate buffer containing 5% D 2 O. The spectrum of hTeloC alone was measured over 1 hour after which 1 mM of CuCl 2 was added and the subsequent spectrum acquired over 2 hours. Finally 1 mM EDTA was added and the spectrum acquired again for 1 hour.

PRELIMINARY EXPERIMENTS
The FRET melting experiments monitor the emission of the donor fluorophore (FAM) as the temperature of the sample is increased. When the DNA is folded, the fluorophores are close together and FAM is quenched; as the temperature increases, the DNA melts, FAM and TAMRA move further apart and FAM is no longer quenched. This results in an increase in the fluorescence of the donor fluorophore FAM; we can use these signals to indicate how much of the sample is folded and also monitor DNA melting.
The initial experiments screened between 10 µM and 100 mM concentrations of cations, added to hTeloC FRET in 10 mM pH 7.4 buffer. A selection of salts were screened, with cations varying in size, charge and geometries: AlCl 3 , CdCl 2 , CoCl 2 , CuCl 2 , Ga(NO 3 ) 3 , HoCl 3 , In(NO 3 ) 3 , S6 FeCl 3 , PbAc 2 , MnCl 2 , HgAc 2 , NiCl 2 , RuCl 3 , SmCl 3 , SrCl 2 , TlAc, TbCl 3 , YbCl 3 , YCl 3 , ZnAc 2 . After the initial screen, several hits were identified which seemed to cause folding at neutral pH at 10 or 100 µM: AlCl 3 , CuCl 2 , Ga(NO 3 ) 3 , HoCl 3 , SmCl 3 , TbCl 3 , YbCl 3 , YCl 3 , HgAc 2 and ZnAc 2 (Fig. S1). At pH 7.4 the majority of hTeloC FRET will be unfolded at ambient temperature and give a high level of fluorescence. As the DNA is normally unfolded at pH 7.4, it is not possible to measure a melting temperature in the absence of the cations however, these cations have induced significantly higher DNA melting temperatures, suggesting they form stable secondary structures.  For the remaining experiments, 50 mM sodium cacodylate was used to enable use of higher concentrations of CuCl 2 . This has two effects though, the buffering capacity increased, but S8 also the concentration of sodium cations, which are known to have a destabilizing effect on i-motif structure. 5 Our initial screen indicated that CuCl 2 was able to fold the DNA at pH 7.4 and the DNA was found to melt at 57°C. Given the DNA is completely unfolded at physiological pH at the start of the experiment (25°C) this indicates a ΔT m of at least +32°C on addition of 10 µM CuCl 2 to 200 nM hTeloC FRET .

DATA FITTING
The sigmoidal curves were fitted to the Hill 1 equation using Origin using the standard constraints at the start and end values: Where is the molar ellipticity at 288 nm, K A is the apparent association constant of [ ] binding Cu 2+ and n is the Hill coefficient.

"COPPER DIFFERENCE" SPECTRA
To investigate how different i-motif forming sequences appear in the "copper difference" spectra sequences from c-Myc, hif-1-α and PDGF-A were also examined in addition to the human telomeric sequence and hairpin control (Fig. S5). Each has a similar shape, consistent with formation of a hairpin-type structure, not an i-motif.

INTRA VS INTERMOLECULAR STRUCTURE FORMATION
The nature of the fast conformational changes indicate formation of an intramolecular structure. 6,7 However, to give an indication of the stability, folding and kinetics of the Cu 2+ complex, UV thermal melting annealing experiments were also performed. The change in absorbance at 295 nm was measured as a function of temperature using different concentrations of DNA (Fig. S5). The absorbance vs temperature profiles for the heating and cooling transitions were found to be non-superimposable both in the absence (Fig. S5) and presence (Fig S6) of Cu 2+ . The hysteresis show that the thermally-induced transitions follow slow kinetics under the experimental conditions. However, they do not change with concentration of DNA (Table S1), indicative of an intramolecular process.

FOLDING KINETICS
To give an indication of the timescale it takes for the structure to turn from i-motif to hairpin, kinetics experiments were performed by mixing DNA and CuCl 2 and monitoring loss of signal at 288 nm in the CD spectra over time. As 10 µM of hTeloC at pH 5.5 showed complete conversion to the alternative structure at 1 mM CuCl 2 , the experiments were performed in triplicate under conditions analogous to the previous experiments (see Fig.   S9). As the concentration of Cu 2+ >> DNA, we considered the conditions as pseudo-first order and the folding data was fitted to a single exponential function as previously described: 8 Where is the ellipticity at 288 nm, t 1 is the characteristic folding time and gives 1/t 1 as the characteristic folding rate. To give an indication of the reverse folding with EDTA, we also investigated the kinetics after addition of folding to hairpin, back to i-motif using EDTA (Fig. S10).

S14
The rates of folding for different concentrations of CuCl 2 were investigated. A summary of the characteristic folding times and rates are given in Table S2.  Fig. 2b). At this point an estimate of the upper limit of the characteristic folding time (t 1 ) can be given as 44 s. The values obtained for the reverse-folding using EDTA were approximately the same within error.
Although these experiments give an insight into how quickly the folding process occurs, the drawback is the dead time of the instrument from addition of the Cu 2+ or EDTA and the first measurements, typically about 8 s. This means that the measurement of the initial rate is not possible. It is also likely that the folding and re-folding processes go via at least one S15 intermediate, but without this initial data, it is not possible to make any firm analysis. To gain full insights into the full kinetics underlying these processes is beyond the scope of this present study; further experiments using stopped-flow would be required.

REVERSIBILITY WITH EDTA
To complement the fitting in Fig 4., we also performed the same analysis with the addition of EDTA to reverse the folding process (Fig. 5). Plotting the molar ellipticity at 288 nm against concentration of EDTA also gave a sigmoidal shaped curve (Fig. S7), fitting this with the Hill1 equation (Eq. S1) gave a Hill coefficient (n) of 5.4 (± 0.7), indicating positive cooperativity (n > 1). Additionally, the half-EDTA concentration for the transition ([EDTA] 50 ) between the two states was found to be 531 (± 24) µM, indicating that an excess of EDTA is required to reverse the folding completely (compared to 382 (± 14) µM for CuCl 2 ). Multiple cycling between each folded structure is possible with sequential additions of CuCl 2 and EDTA ( Fig. 6 and S8), due to the excess EDTA required to return the folded structure to imotif, when sequential aliquots of equimolar amounts of CuCl 2 and EDTA were added, a slight decrease in signal at 288 nm (i-motif) is observed with each cycle.