Spectroscopic analysis reveals the effect of hairpin loop formation on G-quadruplex structures

We study and uncover the effect of hairpin structures in loops of G-quadruplexes using spectroscopic methods. Notably, we show that the sequence, structure, and position of the hairpin loop control the spectroscopic properties of long loop G-quadruplexes, and highlight that intrinsic fluorescence can be used to monitor the formation of non-canonical G-quadruplexes.


Supporting Information
Methods Table S1 Oligo sequences used in this study and their abbreviations, relative fluorescence, and CD peak wavelength. Table S2 Thermodynamic data of oligos used in this study.

DNA Preparation
In this study, DNA oligonucleotides were commercially supplied by Integrated DNA Technologies (IDT). Sequences, as well as abbreviations are shown in Table S1. The oligonucleotides were dissolved to the concentration of 100 μM with ultra-pure nucleasefree water (Invitrogen). Nano-Drop 1000 spectrophotometer (Thermo Scientific) was used to confirm the concentration of DNA oligonucleotides. Oligonucleotides were stored at -20°C before experiment.

Circular Dichroism (CD)
In this study, Jasco J-1500 CD spectrophotometer and Quartz cuvettes with the path length of 1 cm (Hellma Analytics) were used to perform CD spectroscopy. Samples were prepared in a reaction volume of 2 mL in total, containing DNA oligonucleotides with the final concentration of 5 µM, and a reaction salt and buffer of 15 mM KCl or LiCl, and 10 mM LiCac (pH 7.0). Samples were heated at 95 °C in Thermo-Shaker (ALLSHENG) for 5 minutes for denaturation and, thereafter, renatured by cooling to room temperature for around 15 minutes. The samples were excited and scanned between 220 -310 nm at room temperature and spectra were obtained every 1 nm. Accumulation of 2 scans with a 2 s/nm response time were collected and averaged 1, 2 . The data were then normalized and then smoothed over 5 nm 3 . Data was processed and examined with Microsoft Excel.

Fluorescence Spectroscopy
HORIBA FluoroMax-4 and Quartz cuvettes with the path length of 1 cm (Hellma Analytics) were used to perform fluorescence spectroscopy in this study. Samples with a reaction volume of 2 mL in total were prepared, containing DNA oligonucleotides with final concentration of 5 µM, and a reaction salt and buffer of 15 mM KCl or LiCl, and 10 mM LiCac (pH 7.0). Samples were heated at 95 °C in Thermo-Shaker (ALLSHENG) for 5 minutes and then allowed to cool down at room temperature for around 15 minutes. The excitation wavelength of the G-quadruplex containing samples was set at 260 nm and then the emission spectra were collected between 300 -500 nm as reported in previous study 1 . Spectra were obtained every 2 nm at room temperature. The bandwidth was set at 5 nm for both entrance and exit slits. For each oligonucleotide, the data was smoothed over 5 nm. Normalization of spectra was performed relative to the emission intensity of dG3T at 386 nm, i.e. the peak emission of dG3T. dG3T samples were analyzed under the same condition within the same day of the normalized samples. Three independent experiments were performed for each DNA oligonucleotide and results were analyzed with Microsoft Excel. Quantum yield of dG3T in 15 mM K + was obtained with the method previously reported by Sherlock et al 4 .

Thermal Denaturation Monitored by UV Spectroscopy (UV Melting)
Samples containing 5 µM DNA oligonucleotides were prepared in a solution with 15 mM KCl or LiCl, and 10 mM LiCac (pH 7.0) in a reaction volume of 2 mL in total. All samples were denatured at 95 °C for 5 minutes, and cooled down to room temperature for around 15 minutes for renaturation. Agilent Cary 100 UV-Vis Spectrophotometer and Quartz cuvettes with the path length of 1 cm (Hellma Analytics) were used to conduct the UV melting experiments. After loading the sample solutions, the cuvettes were sealed with 3 layers of Teflon tape to prevent vaporization at high temperature. The samples were monitored at 295 nm from 20 to 95 °C with a temperature ramping rate of 0.5 ˚C/min. When temperature reached 95 °C, it was hold for 5 minutes. Following the holding time, a reversed scan at 295 nm was performed with a decreasing rate of 0.5 °C/min until 20 °C. The initial data obtained were corrected by the blank solutions, which contains 15 mM KCl and 10 mM LiCac (pH 7.0) with a reaction volume of 2 mL in total. The data of each DNA oligonucleotide was then smoothed over 11 nm and Microsoft Excel was used to plot its first derivative. The final melting temperature was calculated by averaging the melting temperatures obtained from the forward and reverse scans. For all the G4 samples, enthalpy (ΔHº ) and entropy (ΔSº ) were calculated based on the UV melting curve as described in previous research 5 following equations below, assuming change of folded and unfolded form in specific heat capacity (∆Cpº ) ~ 0. In the equation, θ is the fraction of folded oligos and T is the relative temperature.  Note: Under the condition of 15 mM K + .