Annealing Duplexes to Nanoclews

In order for the nanoclew to act as a stable base for drug delivery, the drug-loaded duplex needs to be able to attach to it. 

In this experiment, we take the preliminary step towards attaching drug-loaded duplexes by assessing the number of single-stranded GI sequences that can be annealed to nanoclews in a w/w ratio through the use of polyacrylamide gel electrophoresis.

Introduction

Due to the high stability of nanoclews under physiological conditions, they serve as ideal carriers of drug-loaded duplexes, which are vulnerable to degradation. In order to achieve this, we designed the nanoclew template to have an overhang binding site (see “Ligation Optimization”) that is complementary to the overhang on the 5’ end of the GI sequence (see “Duplex Formation and Verification”). Therefore, it is expected that the GI sequence would be able to anneal to the nanoclew through simple Watson-Crick base pairing.

Aim

To confirm and optimize the ratio of annealing the GI strand to nanoclews

Methodology

Template and primer were combined with 10x ligation buffer and Milli-Q water to final concentrations of 0.6 uM and 1.2 uM respectively. The sample was then heated to 95C for five minutes and gradually cooled to room temperature over the course of 2.5 hours.

After adding T4 ligase, the samples were each subjected to one of the following treatments:

  • Incubation at room temp for 30 minutes
  • Incubation at room temp for 1 hour
  • Incubation at 37C for 30 minutes
  • Incubation at 37C for 1 hour

These treatments were selected because while a typical ligation involves incubation at room temperature for one hour (or more), [PAPER] has confirmed successful ligation of nicks at 37C after only a 30 minute incubation.

At the designated time point, the samples were heated at 65C for 10 minutes to deactivate T4 ligase. Afterwards, phenol-chloroform extraction was performed to purify the template primer-complex. The samples were then run on a 2% agarose gel to verify circularization

Results

Image 3: Photo of an agarose gel showing the banding patterns made by template-primer complexes that have been subjected to different ligation conditions

As demonstrated by Image 3, while all conditions yielded successfully ligated templates, incubation for only 30 minutes at either temperature resulted in an excess of linear templates. Additionally, the band for one-hour incubation at room temperature is darker than the band for one-hour incubation at 37C. 

Discussion/Conclusion

To minimize the amount of linear template leftover and maximize the amount of circularized template, incubating samples at room temperature for one hour is the optimal protocol (marked by a star in Image 3). This is supported by the band denoting excess linear template being fainter compared to the 30-minute incubation samples, and the band for the ligated template being darker than the one present in the sample that had been incubated at 37C. 

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