Introduction

Image 1: Mechanism of pH-dependent DOX loading and release through changes in i-motif conformation

Aim

To verify the pH-dependent loading and release of DOX by the duplex through the use of an i-motif sequence.

Techniques Used

Ethanol Precipitation of DNA

Ethanol precipitation is a method for concentrating and purifying DNA. It first involves the addition of cationic salts to neutralize the phosphate groups on the DNA backbone, which consequently decreases the polarity of the molecule. Afterwards, ethanol is added which causes the  DNA to precipitate out of the solution [4].

Methodology

Image 2: Visual abstract of testing DOX loading and release

Sample Preparation

Doxorubicin (Sigma Aldrich) was diluted to a stock concentration of 10 mM. For this experiment, it was aliquoted and further diluted to a working concentration of 1000 μM. GI and GcI-based duplexes were diluted to two sets each of 0.25 µM, 0.5 µM, and 1 µM with pH 7.4 IDTE buffer (IDT) for a total of 16 samples. 

After adding DOX working stock to a final concentration of 20 µM in each sample, HCl was added to one set of samples to decrease the pH to 5.5. In summary, there were 8 samples (4 for GI and 4 for GcI) at pH 7.4 in IDTE and 8 samples (4 for GI and 4 for GcI) at pH 5.5. These samples were incubated for approximately 10 hours at 37°C.

Ethanol Precipitation

A salt solution with 2M NaCl and 0.1M MgCl2 was prepared in Milli-Q water, and 100% ethanol was chilled to -20°C. A volume of salt solution equivalent to 10% of the volume of the DOX-DNA solution was added to each sample and mixed thoroughly. Three times this volume of 100% ethanol was added, and the mixture was left to incubate at -20°C for one hour. 

After incubation, the samples were centrifuged at 12,000 rpm for 15 minutes at room temperature. The supernatant was extracted and loaded in triplicates into a 96-well plate to be read by plate reader.

Fluorescence Measurement

Fluorescence was measured at an excitation of 488 nm and an emission of 570 nm using a Varioskan plate reader.

Data Processing

After averaging the triplicate measurements and subtracting the absorbance of blank solutions (75% ethanol, 20% IDTE, 5% salt solution), the fluorescence measurements were expressed as a percentage of the fluorescence of a sample of 20 µM DOX with no DNA added. This amount was subtracted from 100 to yield the amount of DOX loaded.

Results

Image 3: The percentage of DOX bound by GI- and GcI-based duplexes at pH 7.2 and 5.5 (A), with special emphasis on DOX binding by GI duplexes at different pHs (B) and the differential absorption of DOX by GI and GcI at pH 7.2 (C). Error bars represent standard deviation (n=3).

To showcase the behaviour of DOX at different pHs with different duplexes with clarity, the results from the samples containing 1 µM of DNA are presented in Image 3. There are two main features of graph A – the decrease in the amount of DOX loaded onto the GI-based duplexes and the increase in the amount of DOX loaded onto the GcI-based duplexes as the pH decreased (p<0.05, n=3). While the former demonstrates the i-motif’s role in inducing DOX release at pH 5.5, the latter is potentially a subject for further experimentation.

Graph B further emphasizes decreased DOX loading onto the GI with decreasing pH (p<0.05, n=3), and graph C showcases how the presence of the i-motif and its C-G base pair-rich regions enhance the ability of DOX binding for the GI as compared to the GcI at pH 7.2 (p<0.05, n=3). The graphs for all of the data points on the “Raw Data” spreadsheet linked above elucidates the same trend.

Discussion

Based on the decrease in DOX loading onto the GI duplex at pH 5.5 compared to pH 7.2 and the inverse trend seen for GcI, it can be concluded that the presence of the i-motif interferes with DOX intercalation into DNA in acidic conditions. In order to determine the true amount of DOX that is able to be released by the GI under physiological conditions, further experimentation is required. DOX cannot diffuse out of the test tube as it could out of endosomes, so it may, to some extent, re-intercalate within the secondary structures of the GI strand.