Introduction

Image 1: ZnPC loaded onto the G-quadruplex

Aims

• To confirm the ability of ZnPC to be loaded onto the G-quadruplex 
• To evaluate reactive oxygen species generation by ZnPC after laser irradiation

Techniques Used

Scanning Spectrophotometry

Spectrophotometry is a tool that quantitatively analyses the property of materials depending on how much light is absorbed. Scanning spectrophotometry measures the absorbance of light at every wavelength within a range by comparing the difference in intensities between the beam emitted from the machine’s light source and the ray that hits the photodetector after passing through the sample [3].

Singlet Oxygen Sensor Green

Singlet Oxygen Sensor Green (SOSG) is a commercially available reagent composed of fluorescein and anthracene moieties. In its default state, the fluorescein is constantly being quenched by the anthracene. However, in the presence of reactive oxygen species, the anthracene forms an endoperoxide moiety and fluorescence occurs [4].

Methodology

Tris-Acetate Buffer Preparation

Forty millimolar tris-acetate buffer was prepared by first dissolving Trizma acetate salt (Sigma Aldrich) in Milli-Q water. NaCl, KCl, and MgCl2 were added to bring the cation concentrations to 0.1M, 25 mM, and 10 mM respectively. DMSO was added to reach 10% v/v, and the pH was adjusted to 7.4 using acetic acid. 

Sample Preparation

ZnPC (Sigma Aldrich) was dissolved in DMSO to a final concentration of 500 µM. GI and GcI sequences were solubilized in the buffer prepared above to a final concentration of 100 µM. Each sequence was then diluted to concentrations of 0 µM (no template), 2 µM, 10 µM, 20 µM, 50 µM, and 100 µM. The concentration of ZnPC in each sample was 5 µM. Fifty micromolar GI and GcI samples were also created without ZnPC to act as controls.

All samples were run on a UV-vis spectrophotometer (Cary 4000) from 250nm to 750nm using tris-acetate buffer as a blank.

Reactive Oxygen Species Generation

The Singlet Oxygen Sensor Green reagent (Thermo Fisher Scientific) was solubilized in methanol to make a 5 mM stock solution. This was then added to ZnPC or ZnPC and GI samples to a final concentration of 20 nM. A 650 nm laser was used to irradiate each sample for 2 minutes before measuring fluorescence at Ex/Em 504/525 nm using Varioskan.

Results

ZnPC Loading

Image 2: Graph of scanning spectrophotometry results for unbound ZnPC and ZnPC with different concentrations of GI sequence

As shown in Image 2, while ZnPC or GI sequence alone does not absorb at 680 nm, a clear peak forms in samples with both. The height of this peak increases with increasing concentration of DNA, demonstrating that increasing the concentration of G-quadruplexes increases the amount of ZnPC bound and results in a more pronounced bathochromic shift.

Reactive Oxygen Species Release

Image 3: Graph of fluorescence from SOSG as a function of GI concentration

Image 3 demonstrates that the amount of ROS generated generally increases with the amount of G-quadruplex available to bind ZnPC, and that ZnPC alone barely yielded more fluorescence than the blank solution. This is in accordance with our findings that more bound ZnPC results in greater absorbance at 680 nm, since absorbance of red laser radiation is necessary for reactive oxygen species generation.

Discussion

The data gathered supports the claim that ZnPC needs to be loaded onto the G-quadruplex to be effectively used for photodynamic therapy. ZnPC alone does not absorb 680 nm light or efficiently generate ROS but readily does both when combined with the GI sequence. Increasing the amount of GI available for binding also increases the absorbance and improves ROS generation. Further experimentation elucidating the saturation level of ZnPC binding to GI would be helpful in optimizing loading efficiency.