Introduction

Two factors important for duplex formation are buffer composition and annealing temperature. Duplex formation depends upon the presence of cations to reduce electrostatic repulsion between the two negatively charged DNA backbones [1]. After being heated to disrupt secondary structure formation, oligos can be cooled in different ways, which affects the annealing. Cooling can be done by rapid transfer to ice, slow cooling to a low temperature, or cooling to an optimized “annealing” temperature, all of which have varying effects depending on the sequence[2].

For the GI-Nc, we needed to optimize the formation of two different duplexes – the GI and GcI (see sidebar), with the latter acting  as a control for future experiments. Additionally, due to the importance of the GI for drug loading and delivery, further quantitative confirmation of duplex formation was done by performing a fluorophore-quencher assay.

Aims

• To determine the optimal conditions for duplex formation.

• To quantitatively verify formation of the GI-based duplex using a fluorophore-quencher assay.

Techniques Used

Polyacrylamide Gel Electrophoresis

PAGE allows for the separation of macromolecules like DNA based on electrophoretic mobility, which is a function of the size, charge, and conformation of the sample [3]. Additionally, it provides semi-quantitative data regarding the amount of each fragment in the sample based on the brightness of the bands. For this experiment, it was used to evaluate duplex formation under different conditions.

Fluorophore-Quencher Hybridization Assay

Fluorophores are chemical compounds that emit light upon excitation, whereas quenchers decrease the intensity of the fluorescence signal by absorbing and dissipating excitation energy [4]. For this experiment, the FAM fluorophore was conjugated to the 3′ end of the GI and GcI sequences, and the Iowa Black FQ quencher was conjugated to the 5′ end of the sequence complementary to the GI. When duplexes form, the fluorophore and quencher assemble into a nonfluorescent complex, which is a process called “contact quenching”.

Methodology

Overview

Phosphate Buffer Preparation

A stock solution of 0.1M pH 7.2 phosphate buffer was prepared by adding 0.1M sodium phosphate monobasic solution to 0.1M sodium phosphate dibasic solution until the desired pH of 7.2 was reached. NaCl, MgCl2, and KCl were dissolved in this buffer to final concentrations of 10mM K, 0.8mM Mg, and 20mM Na. 

DNA Resuspension

Two stock solutions of each of the four DNA sequences (GI, GcI, and their complementary sequences) were created: 200 µM in TE buffer, and 200 µM in phosphate buffer. DNA was resolubilized by resting at room temperature for one hour.

Duplex formation

Duplexes were synthesized by combining GI, and GcI sequences with their respective complementary sequences in equal amounts. Annealing was tested in either TE or phosphate buffer. Three different protocols were tested for each:

• Room Temperature: Incubation at room temperature for 10 minutes
• Normal: 95°C for 5 minutes > 4°C
• Annealing: 95°C for 5 minutes > Annealing temperature (53.7°C for GI and 35.5°C for GcI, calculated via IDT) for 2 minutes > 4°C

Polyacrylamide Gel Electrophoresis

A 20% PAGE gel was prepared accordingly:

Samples were diluted with 6x loading dye, and 40 ng of DNA was loaded into each well. A low molecular weight DNA ladder (Thermo Fisher) was loaded alongside the samples. The gel was run at 150V and 40 mA for two hours before being stained with SYBR Gold.

DNA Hybridization Assay

The GI and GcI sequences were modified with a 3’ FAM and the GI complementary strand was modified with a 5’ Iowa Black FQ quencher (all IDT). All DNA sequences were diluted to a stock concentration of 100µM with IDTE buffer.

The following samples were prepared:

• 1 µM GI alone to measure innate fluorescence without quenching
• 1 µM GI + 1 µM non-quenching GI complementary to assess the influence of the presence of complementary sequences on fluorescence
• 1 µM GcI + 1 µM quenching GI complementary to analyze the effect unbound quenchers have on fluorescence
• 1 µM GcI + 1 µM non-quenching GI complementary to assess the influence of the presence of non-complementary sequences on fluorescence
• 1 µM GI + 1 µM quenching GI complementary to test duplex formation
• Nuclease-free water to serve as a blank

The samples were heated to 95°C for 5 minutes and cooled to 4°C to facilitate annealing. The fluorescence was then measured on Varioskan Flash at excitation/emission wavelengths, 495/520 nm.

Results

Duplex Formation Optimization

Image 1: Photos of polyacrylamide gels testing GI (left) and GcI (right) duplex formation under different conditions

Formation of the duplex is characterized by a slight upwards shift of the band compared to either the GI/GcI or their complementary sequences alone. In Image 1, all lanes with duplexes have two bands – a lower band corresponding to excess complementary strands, and an upper band higher than the GI/GcI sequence demonstrating successful duplex formation under all conditions tested.

Duplex Formation Verification

Image 2: Graph comparing fluorescence with different sequences

Image 2 shows a significant decrease in fluorescence signalling of the “GI + quenching complementary” compared to GI alone,  “GI + non-quenching complementary”, and “GcI + quenching GI complementary” (all n=5, p=0.05). This demonstrates that the extent of quenching for the “GI + quenching complementary” sample cannot be attributed solely to the presence of quenchers or additional non-fluorescent DNA, and is the result of successful duplex formation.

Discussion

Through the use of PAGE, we have confirmed the successful formation of GI- and GcI-based duplexes using both phosphate and TE buffer and using all three (room temperature, “normal”, and “annealing”) annealing protocols. Based on our data, all conditions were equally effective. With the fluorophore-quencher hybridization assay, the formation of GI-based duplexes was verified and quantified. As a result, a significant decrease in fluorescence signal due to contact quenching was seen.