DNA based multi-copper ions assembly using combined pyrazole and salen ligandosides

The pyrazole and salen ligandosides, when combined, are able to create stable multi-copper ion complexing DNA duplex structures in a cooperative fashion.

The purified fractions were concentrated, desalted on Waters Sepac-C18 cartridges and concentrated again.

Sample preparation and melting experiments
Melting profiles were measured on a JASCO V-650 spectrometer using quartz glass cuvettes with 10.00 mm path length. The samples contained 150 mM NaCl, 10 mM CHES buffer and 1 μM of each strand in a final volume of 200 μL. For strands 1a/1b, 2a/2b, 3a/3b, and 4a/4b the oligonucleotides with Cu 2+ were hybridized by slowly cooling down the samples from 90 °C to room temperature overnight. Each sample was prepared and measured at least three times in parallel. The solutions were covered with silicon oil and tightly plugged. UV Absorbance was recorded in the forward and reverse direction at temperatures from 20 °C to 80 °C with a slope of 1 °C/min. Three denaturing and renaturing ramps were performed for each sample and averaged for evaluation of the melting point. TM values were calculated as the zero-crossing of 2 nd derivate of the 349 nm background corrected change in hyperchromicity at 260 nm.

UV / CD spectra measurements
UV and CD spectra were measured on a JASCO V-650 or J-810 spectrometer using quartz glass cuvettes with 10.00 mm path length. For the strands with mixed salen and pyrazole bases, 5a/5b, 6a/6b and 7a/7b, the duplex (3 μM of each strand) was first reannealed in 150 mM NaCl, 10 mM CHES buffer without Cu 2+ ions or ethylenediamine overnight. Then an excess of ~30 fold ethylenediamine, diluted in degased Milli-Q water, was added and the solution was incubated at 4 °C overnight. Finally, different amounts of Cu 2+ were added into individual samples and final volume was constant (200 μL). After Cu 2+ was added, the samples were kept at 4 °C and measured in 2 h. Each sample was prepared and measured at least twice in parallel at 20 °C. For UV measurements, five spectra were accumulated. For CD measurements, ten spectra from 320 nm to 210 nm were accumulated with scanning speed of 500 nm/min at 20 °C. Blank correction was made of aqueous solution of buffer and salt and measured for each series of titration measurement.

ESI Mass measurements
For duplex 5a/5b, 30 μM of duplex was hybridized in 150 mM NH4OAc as above. Here, no CHES buffer and NaCl aq. were used. After addition of ethylenediamine, the mixture was incubated at 4 °C overnight, then Cu 2+ (3 eq.) was added. Before ESI measurement, a mixture (10 μL) of imidazole (250 mM) and piperidine (250 mM) in 80 % aq. acetonitrile was added to the sample (40 μL).

Fig. S1
Summary of reported metal base pairs. Backbone part is not shown here and only one kind of metal ion is shown.

II.
Selected thermodynamic data and spectroscopy data for melting curves and CD experiments [a] These values were determined by van't Hoff plots from the melting profiles. Conditions: 150 mM NaCl, 10 mM Na2HPO4/NaH2PO4 buffer pH 6.0 / 7.4 or CHES buffer pH 9.0, 1 μM oligonucleotide, with or without 1 μM Cu 2+ , final volume of 200 μL.
The mixture was shaken for 24 hours at 4 °C, followed by the extraction with diethyl ether (3 × 4 mL), dried over MgSO4, filtered and concentrated. The crude product was dissolved in heptane and 2-propanol (v/v 99/1) and analyzed by chiral HPLC directly.

HPLC analysis:
Schimadzu where PA1 and PA3 are the HPLC peak areas of 1 and 3, respectively, and f is the correction factor determined to be 0.69 from the fitting curve (Fig. S17).