Introduction

Nanoclews resist degradation when exposed to nucleases, diluted to low concentrations, or are in human serum.  This property makes them suitable for drug delivery in vivo [1]. When GI-based duplexes are annealed to a nanoclew, a stable structure capable of delivering a concentrated dose of therapeutics with high specificity is created.

To facilitate conjugation, the nanoclew template contains an overhang binding site complementary to the overhang sequence on GI-based duplexes. We chose to examine binding efficiency using a w/w ratio because it is important to determine the saturation level of duplex binding to nanoclews in order to create GI-Nc complexes with a consistent amount of drugs loaded onto each one.

Aim

To determine the most efficient w/w ratio for annealing GI-based duplexes to nanoclews.

Methodology

Nanoclews produced by RCA for a duration of  6 hours were prepared according to “Sizing and Characterization” and resuspended in IDTE buffer. GI-based duplexes were synthesized in IDTE buffer by heating to 95°C for 5 minutes and then cooling to 4°C in a thermocycler. Both were then Nanodropped to determine the concentration of DNA in nanograms per microliter.

Samples with the following nanoclew:duplex w/w ratio were prepared: 0:1, 0.5:1, 1:1, 2:1, 5:1, 10:1, 15:1, and 1:0. 100 ng of the duplex were in each reaction tube, and the amount of nanoclew added depended on the w/w ratio. The samples were topped up with Duplex Buffer (IDT) to the same volume.

To anneal, the samples were heated to 95°C for 5 minutes, then incubated at room temperature for three hours. 

An 8% polyacrylamide gel was prepared accordingly:

Samples were loaded with a 100 bp DNA ladder (NEB) and run at 150V, 40 mA for 30 minutes. The gel was then stained with Sybr Gold and imaged using the MultiImage Light Cabinet and AlphaImager EC.

Results

Image 1: Polyacrylamide gel demonstrating the annealing of GI sequences to nanoclews at difference ratios

Annealing of duplexes to the nanoclew is evaluated by assessing the duplex’s band intensity on a polyacrylamide gel. The nanoclew is too large to travel down a polyacrylamide gel so it will remain at the top, along with any duplexes bound to it. Since the amount of duplexes in each sample is the same, band intensity is solely determined by the amount of free duplex. Looking at Image 2, a gradual lightening of the band denotes that the duplex is apparent as the concentration of nanoclews increases. At a nanoclew:duplex ratio of 5:1, the duplex’s band disappears, signifying that all duplexes have been annealed.

Discussion

Through the use of PAGE, we have verified that duplexes are capable of annealing to nanoclews, and that, at a nanoclew:duplex w/w ratio of 5:1, all duplexes are bound. In accordance with expectations, increasing the amount of nanoclew steadily increased the amount of bound duplex. These results validate the sequence design for the nanoclew and duplex and demonstrate the viability of using the nanoclew as a vehicle for stabilizing and transporting drug-loaded duplexes.