Circular Dichroism Experiments

All CD experiments were performed at 25 °C in 10 mM K2HPO4 buffer containing 80

mM KCl and 1 mM EDTA. CD spectra were recorded using a Jasco J-810 spectrophotometer with a 1-cm pathlength quartz cell at a scan speed of 50 nm/min and response time of 1 second. Appropriate amount of compounds were sequentially titrated from the stock solution into the DNA solution in the cuvette until the desired mole ratios of compound to quadruplex were obtained. The spectra were averaged over four scans. A buffer baseline scan was collected in the same cuvette and subtracted from the average scan of each ratio. Data were processed and plotted using Kaleidagraph 4.0 software.

4.2.3 Thermal Melting Studies

Thermal denaturation studies were conducted on a Cary 300 BIO UV-visible spectrophotometer in quartz cuvettes of 1 cm pathlength. Compound-DNA solutions were prepared in low salt buffer containing 10 mM TRIS buffer (pH 7.4), 10 mM KCl and 1 mM EDTA. The absorbance of the oligonucleotides was monitored at the recommended wavelength of 295 nm for quadruplex sequences and 260 nm for duplex sequences as a function of temperature, and DNA without compound was used as a control. Samples of compound to DNA ratios ranging from 0:1 to 4:1 were prepared. Cuvettes were mounted in a thermal block, and the solution temperatures were monitored by a thermistor in a reference cuvette with a computer-controlled heating rate of 0.5 °C/min. Experiments were generally conducted at quadruplex concentrations in the range of 2-3 µM in TRIS buffer containing 10 mM KCl. Data were analyzed and plotted using Kaleidagraph 4.0 software.

4.2.4 Surface Plasmon Resonance Studies

Biosensor SPR experiments were performed with a four-channel BIAcore 2000 optical biosensor system (BIAcore, Inc.) and streptavidin-coated sensor chips (BIAcore SA with linked streptavidin). All DNA samples, for either duplex- or quadruplex-binding experiments, were

used as single strands to prevent dissociation in the SPR flow system. The chips were prepared for use by conditioning with a series of 1 min injections of 1 M NaCl in 50 mM NaOH followed by extensive washing with buffer. 5'-Biotinylated DNA samples (25-50 nM) in HBS buffer were immobilized on the flow cell surface by non-covalent capture as previously described [26]_{. Three flow cells were used to immobilize DNA samples, and the first flow cell}

was left blank as a control. Interaction analysis was performed by using steady-state methods with multiple injections of increasing compound concentrations over the immobilized DNA surface at 25 ºC. Biosensor experiments were conducted in filtered, degassed HEPES buffer (10 mM HEPES, 100 mM KCl, 3 mM EDTA, 0.005 v/v of 10% P20 BIACORE surfactant, pH 7.3) at 25 °C. Flow cell 1 was left blank as a reference, while flow cells 2-4 were immobilized with DNA on a streptavidin-derivatized gold chip (SA chip from BIAcore) by manual injection of DNA stock solutions (flow rate of 1 µL/min) until the desired value of DNA response was obtained (350-400 RU). Compound solutions were prepared with the running buffer by serial dilutions from stock solution. Typically, a series of different ligand concentrations (1 nM to 10 µM from 20 mM H2O stock) were injected onto the chip (flow rate of 50 µL/min, 5-10 min)

until a constant steady-state response was obtained followed by a dissociation period (buffer, 10 min). After every cycle, the chip surface was regenerated (20 s injection of 10 mM glycine solution, pH 2.0) followed by multiple buffer injections.

The instrument response (RU) in the steady-state region is proportional to the amount of bound drug and was typically determined by linear averaging over a 10-20 s or longer time span, depending on the length of the steady-state plateau. The predicted maximum response per bound compound in the steady-state region (RUmax) was determined from the DNA molecular weight, the amount of DNA on the flow cell, the compound molecular weight, and the refractive index gradient ratio of the compound and DNA, as previously described [27]_{. In}

pointing to more than one binding site in these DNA sequences. The number of binding sites was estimated fitting plots of RU versus Cfree. These methods can also be used to determine an

empirical RUmax value. The RUmax value is required to convert the observed response (RU) to the standard binding parameter r (moles of drug bound per moles of DNA hairpin)

r = RU/RUmax

which is useful for comparison of a compound binding to different DNAs to obtain the binding constants, the data were evaluated with different interaction models to obtain an optimal fit using BIAevaluation (BIAcore Inc.) and Kaleidagraph (Synergy Software) software for nonlinear least-squares optimization of the binding parameters:

One site: r = (K1Cfree)/(1 + K1Cfree)

Two site: r = (K1Cfree + 2K1K2Cfree2)/(1 + K1Cfree + K1K2Cfree2)

Three site: r = (K1Cfree + 2K1K2Cfree2 + 3K1K2K3Cfree3)/(1 + K1Cfree + K1K2Cfree2 +

K1K2K3Cfree3)

where K1, K2 and K3 are equilibrium constants for three types of binding sites and Cfree is the

concentration of the compound in equilibrium with the complex and is fixed by the concentration in the flow solution.

4.2.5 Nuclear Magnetic Resonance Studies

Quadruplex DNA samples were prepared in degassed phosphate buffer, containing 80 mM KCl, 10 mM K2HPO4 and 0.1 mM EDTA, and reconstituted in 90% H2O:10% D2O. 0.01

mM DSS was employed as an internal reference. DNA concentrations were in the range of 0.1 mM to 0.3 mM unless otherwise mentioned. The final DNA samples were adjusted to pH 7.0 using 1M HCl or 1M KOH solutions. Finally, the NMR samples were heated past their transition temperature and annealed to room temperature several times before collecting the spectra. Experiments were performed on a Varian Unity 600 spectrometer. DB compounds were titrated into the quadruplex DNA with compound to DNA ratios varying from 1 to 3.

Temperature-dependent 1_{H-spectra were recorded from 15 ºC to 45 ºC using jump-return and}

WATERGATE methods for solvent suppression [28]_{. All NMR data were processed and analyzed}

with a combination of VNMR (Varian Inc.), NMRPipe (NIH) and MNova (Mestrelab Research) software.

4.3 RESULTS AND DISCUSSION