Identification of new DNA i-motif binding ligands through a fluorescent intercalator displacement assay† †Electronic supplementary information (ESI) available: Experimental, supporting fluorescence and UV-Vis data, Job plot, binding curves, CD spectra, melting temperatures and SPR data. See DOI: 10.1039/c7ob00710h

This work describes a new way to screen for i-motif binding compounds and several new families of ligands with potential for use in experiments into the structure and function of i-motif DNA.


EXPERIMENTAL PROCEDURES 1) 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 10 mM sodium cacodylate buffer at the respective pH. Annealed samples were thermally annealed in a heat block at 95°C for 5 minutes and cooled slowly to room temperature overnight. Candidate FID probes acridine orange, thiazole orange and crystal violet were purchased from Sigma Aldrich. Ethidum bromide and 1-pyrenemethanol were purchased from Fisher Scientific. Stock solutions of ligands at 10 mM were made in DMSO and were stored at -20°C, subsequent dilutions were made in the appropriate buffer. The Gen-Plus library from Microsource Discovery Systems Inc. consisting of 960 drug standards with approval in Europe, Japan or the USA was supplied as 10 mM solutions in DMSO which were diluted to 1 mM in 96 well plates. Data manipulation was performed using Origin 8.0.

2) FLUORESCENCE SPECTROSCOPY
Fluorescence spectra were measured on a Horiba Jobin Yvon Fluorolog a Perkin-Elmer LS-55 fluorescence spectrometer with a Starna Scientific type 28/9-F 1 cm path length quartz cuvette. All experiments were performed in triplicate. DMSO stock solutions of the probes were diluted to 2.5 μM in buffer at the desired pH. hTeloC (5′-d[TAA-CCC-TAA-CCC-TAA-CCC-TAA-CCC]-3′) DNA samples were prepared at 500 μM in buffer at the respective pH. In the experiments, 200 μL of probe (2.5 μM in buffer) was aliquoted into a cuvette and excitation and emission spectra of the ligand-DNA complex were measured. Then, 1 μL of the prepared solution of DNA (2.5 µM) was added to mimic the 1:1 binding event. After mixing and allowing 5 minutes to equilibrate, excitation and emission spectra of the ligand-DNA complex were measured again.
Fluorescence enhancement measurements using TO were performed using TO diluted in the respective buffer. DNA samples of hTeloC and c-MYCC (5′-d[TCC-CCA-CCT-TCC-CCA-CCC-TCC-CCA-CCC-TCC-CCA]-3′) were diluted to 100 μM in buffer at the respective pH and annealed. In the experiments, 200 μL of TO (5 or 2.5 μM) was added to the cuvette and an emission spectrum was taken to observe the fluorescence in the buffer conditions in the absence of DNA. DNA was titrated into the sample, mixed, allowed to equilibrate for 5 minutes after each addition, then excited at 430 nm; fluorescence emission spectra were measured from 440 to 650 nm.

3) UV-VIS SPECTROSCOPY
Samples were measured using on an Agilent Technologies Cary 60 UV-Vis spectrophotometer with a Starna Scientific 1 cm path length quartz cuvette over a range of 350-550 nm at a rate of 300 nm/s with a data interval of 0.5 nm with a dual beam at room temperature. Samples containing only buffer were measured and subtracted from the data. UV-Vis titrations to determine binding affinity were performed using hTeloC (5 µM) in buffer at pH 5.5. A concentrated stock of TO prepared in buffer at pH 5.5 was prepared for titration into 200 µL of hTeloC to give final TO concentrations of 0, 1. 25, 5.2, 3.75, 5, 7.5, 10, 15 and 20 µM. An analogous titration without DNA was subtracted from the data. Binding data were fitted using non-linear regression.

4) CIRCULAR DICHROISM SPECTROSCOPY
hTeloC (10 µM) was diluted in buffer at pH 5.5, pH 6.0 and pH 7.4 to give a total volume of 200 µL for each sample and were annealed overnight. Spectra were recorded using a Jasco J-810 spectropolorimeter with a Starna Scientific type-21 quartz cuvette of 1 mm path length. Scans were performed at 20°C from 220 nm to 320 nm with a scanning speed of 200 nm/min, a response time of 1 s, pitch of 0.5 nm and a bandwidth of 2 nm. Blank samples containing only the respective buffer were recorded and subtracted from the data. For each sample, TO or tobramycin was titrated directly into the cuvette containing the DNA and solutions were thoroughly mixed before recording spectra.
All spectra show an average of three scans. TAT-AGC-TAT-A)-TAMRA-3′); donor fluorophore FAM is 6-carboxyfluorescein; acceptor fluorophore TAMRA is 6-carboxytetramethyl-rhodamine; were prepared as a 400 nM solution in buffer at the respective pH and then annealed. Strip-tubes (QIAgen) were prepared by aliquoting 10 μL of the annealed DNA, followed by 10 μL of TO solutions made in the same buffer. Control samples for each run were prepared without TO. Fluorescence melting curves were determined in a QIAgen Rotor-Gene Q-series PCR machine, using a total reaction volume of 20 μL. Samples were held at 25°C for 5 minutes then ramped to 95°C at increments of 1°C, holding the temperature at each step for 1 minute.

5) FRET MELTING EXPERIMENTS
Measurements were made with excitation at 470 nm and detection at 510 nm. DNA melting points were determined using the first derivative of the melting curve, any experiments where the inflection point was not able to be determined (i.e. the transition does not occur before the end of the experiment) were defined to have a T m of >95°C. Final analysis of the data was carried out using QIAgen Rotor-Gene Q-series software and Origin or Excel.

6) FID ASSAY
TO was diluted to 2 µM in buffer at pH 5.5 and hTeloC i-motif was diluted to 50 µM. The tested ligands were diluted to 50 µM in buffer at pH 5.5. 196 µL of the TO solution was excited at 430 nm and the background fluorescence recorded from 450 to 650 nm. The background fluorescence emission intensity at 450 nm was normalised as 0%. Then 4 µL of hTeloC was added, mixed, allowed to equilibrate for 5 minutes and a second background fluorescence spectrum was taken when the sample was excited at 430 nm. The fluorescence emission intensity at 450 nm was normalized as 100% fluorescence. Then 1 µL aliquots of ligand were titrated into the sample and a spectrum measured. TO displacement was calculated using Equation S1 and plotted against concentration to calculate the DC 50 .

Equation S1
= 1 - The IM-FID assay was conducted using 384-well microplates (Corning® Low Volume 384 well Black Flat Bottom Polystryrene NBS TM Microplate) at 25°C. Microplate wells were filled with 40 μL of a testing solution consisting of hTeloC (0.5 μM) and TO (1.0 μM) in buffer at pH 5.5. Then 0.5 μL of library compound solution (1 mM in DMSO) was added into each well. Each plate had three control wells of DNA in buffer and another three reference wells of DNA and TO in buffer without ligand. After mixing, plates were read on a BMG CLARIOstar using an excitation filter from 400 to 430 nm and an emission filter from 460 to 480 nm. Each scan was performed three times. The basal fluorescence signal (F c0 ) were assigned as the average fluorescence intensity read from the control wells. The 100% fluorescence intensity read (F reference ) was assigned as the average fluorescence intensity read from reference wells. The DC 50 for each compound was calculated using the average of three reads (F read ) using Equation 1. Hit compounds were ranked according to DC 50 (Supporting information).

7) SURFACE PLASMON RESONANCE
SPR experiments were performed using a GE Healthcare Biacore T200 instrument with a series S streptavidin (    Where:  = the fraction of binding measured; K 1 = the equilibrium association constant for the first binding site; K 2 = the equilibrium association constant for the second binding site. The results from the binding studies support the model that two thiazole orange molecules bind to one i-motif.   Figure S15: TO displacement versus concentration plot for mitoxantrone (red), tilorone (blue), tobramycin (green) and tyrothricin (purple) using hTeloC (1 µM) TO (2 μM) and ligand (0-5 µM) at pH 5.5 in 10 mM sodium cacodylate buffer.