Electronic Supporting Information for "Metal-triggered Topology Switching in Bipyridine-modified DNA G-Quadruplexes"

All data names are either directly linked to the individual samples with their respective names, which are explained in the manuscript and the supporting information, or the IUPAC names of the respective compounds. CD data has been saved as either Excel-workbook files (.xlsx) or .csv to enable direct analysis of the raw data with any analysis tool. We have separated the CD data into 3 sections.

The first and main one covers all topology analysis files. The second one under the subfolder "PEG200 Experiments" contains CD measurements performed under molecular crowding condition with polyethylene glycol 200 (PEG200). The third one under the subfolder "Melting Data" contains the thermal denaturation experiments associated with the CD experiments.

The raw data associated with the kinetic experiments discussed in the manuscript can be found under "Kinetics" and are divided into the 4 temperatures which measurements have been performed at, all saved as .csv files to ease access and freedom of choice of the analysis program. Molecular dynamics (MD) simulations shown and discussed within the manuscript can be found in the folder "MD". The associated data was packaged into the individual structures.

The MD movies resulting from the calculations can be displayed using open access software such as ChimeraX 1.9. Fluorescence microscopy measurements can be found in the folder "Microscopy". All FRET-based measurements can be found under the subfolder "Confocal->FRET".

Each measurement is divided into 3 channels (brightfield, red and green channel) which is indicated with the suffix ch00, ch01 or ch02 respectively within the nomenclature. "U2OS" denotes the name of the cells used in this experiments which is explained in detail in the manuscript. The presence or absence of "Ni" indicates whether the measurements were performed in presence or absence of Ni2+ as metal source. The experimental details are explained in the manuscript.

In the subfolder "Transfection" the measured green fluorescence count of each sample and are divided into samples measured in U2OS or HeLa cell lines as denoted in the file name in addition to the standard sample nomenclature. The data is presented as .csv. In the further subfolder "MTS Assay" we have included the data of the cell viability assays, as discussed in the manuscript.

The measurements are divided into cell line (HeLa or U2OS) and sample. The mass spectrometry data can be found in the folder "MS". Individual measurements are packaged into subfolders of the samples.

These measurements were obtained on a Bruker timsTOF mass spectrometer and are saved in a ".d"-folder file. In order to analyze this data, proprietary software such as "Bruker Compass DataAnalysis" or equivalent is needed. Please download the entire folder found under each subfolder denoting sample name (example: htel22_L2a -> htel22-L2a.d) NMR raw data can be found in the folder "NMR".

Here the three novel structures and their respective NMR spectra have been saved into separate folders denoted with the IUPAC names of the chemical compounds as detailed in the manuscript. The individual folder structure is identical to the data directly obtained from the Bruker NMR spectrometers. We advise to analyze the data in the open-access software of Bruker "TopSpin 5".

METHODOLOGICAL INFORMATION: 1H-NMR-Spectra were obtained using Bruker AV 400, Bruker AV 500 Avance NEO / Agilent DD2 und Bruker AV 600 spectrometers manufactured by Bruker Physics and were carried out in deuterated chloroform (CDCl3), deuterated acetonitrile (CD3CN), deuterated dimethyl sulfoxide (DMSO-d6), deuterated tetrahydrofuran (THF-d8), deuterated methanol (MeOD), deuterated acetone (Aceton-d6) and deuterated dimethylformamide (DMF-d7).

The chemical shifts 𝛿 were determined relative to a standard (trimethylsilane, 𝛿 = 0 ppm) in ppm. All 1H-NMR-spectra were calibrated on the residual proton signal of the respective deuterated solvents (CDCl3: 𝛿 = 7.26 ppm, CD3CN: 𝛿 = 1.93 ppm, DMSO-d6 = 2.49 ppm). The coupling patterns were denoted with the following terms: s (singulet), d (doublet), t (triplet), m (multiplet). 13C-NMR-Spectra were obtained using Bruker AV 400, Bruker AV 500 Avance NEO / Agilent DD2 und Bruker AV 600 spectrometers manufactured by Bruker Physics and were carried out in deuterated chloroform (CDCl3), deuterated acetonitrile (CD3CN) and deuterated dimethyl sulfoxide (DMSO-d6).

The chemical shifts 𝛿 were determined relative to a standard (trimethylsilane, 𝛿 = 77.0 ppm) in ppm. All 1H-NMR-spectra were calibrated on the residual 3 proton signal of the respective deuterated solvents (CDCl3: 𝛿 = 77.16 ppm, CD3CN: 𝛿 = 1.3, 117.7 ppm, DMSO-d6 = 39.7 ppm). For standard CD measurements, samples were prepared in the same way as for the UV-VIS-based thermal denaturation studies.

CD spectra were measured on an Applied Photophysics Chirascan qCD spectropolarimeter (350 – 205 nm, 1.0 s time-per-point, step size 1 nm, bandwidth 0.5 nm, 3 repeats) at 25 °C unless otherwise indicated. Temperature was controlled using a Quantum Northwest temperature control attached to a sample probe. The background was measured in the same cuvette as the sample.

All spectra were averaged, background corrected (cuvette, buffer and electrolyte), smoothed (Savitzky-Golay, window size 5) and zeroed to the signal at 350 nm. Further details can be found in the SI. For the kinetic measurements by CD spectroscopy, the samples were prepared in the same way as for the UV-VIS-based thermal denaturation studies.

The CD signal at 260 nm was chosen as this was indicative of the formation of an antiparallel G-quadruplex topology formed from the hybrid topology. The sample was kept at the desired temperature by the temperature control with an in-sample thermometer. At t = 0 s, 4.4 μL of 1 mM metal salt solution was added to the DNA sample and quickly mixed by pipetting.

The measurement intervals were 15 s over a period of up to 20000 s. The recorded CD intensities were then plotted over time and fitted with an exponential decay curve which was used to calculate the concentration of the G-quadruplex without any metal (educt). To assess the order of this reaction we plotted the concentration of the G-quadruplex without any metal bound on a logarithmic scale over the recorded time.

This was repeated for each metal cation (Cu2+, Ni2+, Zn2+) and over four temperatures (284 K, 289 K, 298 K, 306 K). Out of the corresponding kinetic rate constants an Eyring plot was created to extract the enthalpy (ΔH‡) and entropy (ΔS‡) of activation as well as the activation energy at each temperature (Ea or ΔG‡). MD simulations were carried out as described and cited in the manuscript using the Gromacs 2019.2 program with the AMBER force field ff99bsc1 or the nucleic acid parts.

A detailed explanation can be found in the SI. U2OS and HeLa cells were plated (1x105 cells per well, 200 𝜇L, 0.8 cm2) 24 hours before each experiment and grown in DMEM media (Gibco) supplemented with 10% FBS. To measure cellular toxicity of the samples, MTS assays were conducted using the Promega CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit, following manufacturer’s instructions.

Cells were incubated with DNA samples over 6 hours at varying concentrations from 5-2000 nM. Absorbance of the MTS reagent was then measured at 490 nm. The experiment was conducted in triplicate.

Confocal images were acquired using a Leica SP5 II confocal microscope after incubation of U2OS/HeLa cells with the DNA samples for 16 hours. Images were taken with a 100× oil immersion objective (correction collar, NA = 1.2, Leica) after excitation using an internal Ar+ laser at 495 nm and detection at 500-700 nm. DNA transfection was performed with lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions: dye-labelled G4 DNA was incubated with lipofectamine at a 3:1 ratio of lipofectamine:DNA in Optimem media (Gibco) for 15 min.

The lipofectamine/DNA mixture was then incubated with cells for up to 24 hours and imaged every 4h using a Sartorius Incucyte S3 Live-Cell Analysis System from which the total green count of the selected cells was plotted over time to determine the optimal incubation time after which no more signal enhancement was present. All cell imaging experiments are an average of at least two independent biological repeats. Instances where images of three biological repeats were used are highlighted in relevant figure captions.

To measure cellular toxicity of the probe, MTS assays were conducted with a Assay kit reagents mix consisting of a MTS solution in MilliQ water at 2 mg/mL and a phenazine methosulfate (PMS) solution in MiliQ water at 0.92 mg/mL which were mixed in a 2:0.1 ration (MTS:PMS). This solution was then mixed with DMEM containing 10% FBS in a 1:4 ratio (MTS/PMS mix:medium). HeLa and U2OS cells were grown in high glucose Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO2 in humidified air at 5000 cells per well in a 96-well plate over 16h after which media was replaced with fresh media and incubated with the respective oligonucleotide compound and again incubated under identical conditions for 16h.

Lastly media was removed and replaced with MTS reagent/buffer mixture and incubated for 4h. Absorbance of the MTS reagent mix was then measured at 490 nm from which absorbance at 630 nm was subtracted as background correction. The experiment was conducted in triplicate.

Positive controls were conducted with DMSO to ensure no dehydrogenase activity. Negative controls were conducted with cells in just medium to ensure full dehydrogenase activity. The respective absorbance maximum and minimum absorbance values were then normalised to the negative and positive controls.

DNA transfection was performed with lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions: G4 DNA was incubated with lipofectamine at a 3:1 ratio of lipofectamine:DNA in DMEM (Gibco) for 15 min. The lipofectamine/DNA mixture was then incubated with cells for up to 24 hours and imaged every 4h using a Sartorius Incucyte S3 Live-Cell Analysis System from which total green count over all cells within each well was plotted over time to determine the optimal incubation time after which no more signal enhancement was present.

All cell imaging experiments are an average of at least three independent biological repeats. Confocal images of single fluorescently labelled G-quadruplex strands (FAM label) were acquired using a Leica SP5 II confocal microscope after incubation of U2OS/HeLa cells with the DNA samples for 16 hours. Images were taken with a 100× oil immersion objective (correction collar, NA = 1.2, Leica) after excitation using an internal Ar+ laser at 495 nm and detection at 500-700 nm.

For FRET based experimets, confocal images of U2OS and HeLa cells in 8 well chamber slides were acquired using a Leica Stellaris 8 inverted microscope equipped with a diode laser (405 nm) and a white light laser (440-790 nm). Images were collected using a 63x oil immersion objective (correction collar, NA = 1.4, Leica) at a resolution of 512 x 512 pixels. Samples were excited using the white light laser at 495 nm, and emission detected 505-650 nm, using Airy pinhole 1.