Reactive Oxygen Species Generation

In order to serve as the template for nanoclew synthesis, single-stranded linear DNA must be processed to form a circular template-primer complex.

In this experiment, we first closed the linear template into a loop by annealing the primer to it. To seal the nick in the template, we varied the temperature and incubation time of the ligation step to determine the most efficient method for circular complex synthesis.


Nanoclew synthesis begins with the formation of a circular template-primer complex, which is used by phi29 DNA Polymerase for the rolling circle amplification (RCA) process. During RCA, the polymerase repetitively amplifies the complex, synthesizing a length of ssDNA that folds into the nanoclew.

Our template design is based on the work of [SIRNA PAPER]. It incorporates an overhang binding site for the annealing of the aptamer and drug-loaded duplexes to the nanoclew. This binding site is flanked by palindromic sequences that direct nanoclew folding. Additionally, on either end of the template are regions complementary to the primer, which enables for initial loop formation.

Image 1: Linear (top) and circularized (bottom) nanoclew template with the overhang binding site in yellow, palindromic sequences in orange, and primer binding site in green


To optimize the synthesis of the circular template-primer complex by varying the temperature and incubation time of the ligation reaction

Techniques Used

Agarose Gel Electrophoresis

Agarose gel electrophoresis allows for the separation of macromolecules like DNA based on size and 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 determine the efficiency of and confirm template circularization under each condition.

Phenol-Chloroform Extraction

Phenol-chloroform extraction is a liquid-liquid extraction technique for purifying DNA samples. While liquids partition into the lower organic phase and proteins are in the interphase, DNA can be isolated from the upper aqueous phase. As a result, we used this technique to purify our circularized template-primer complexes.


Image 2: Visual abstract of ligation optimization process


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


Template-Primer Annealing:

Template and primer (IDT) were diluted to master stocks of 100 uM using pH 7.5 IDTE Buffer. Working stocks of 10 uM of both were then made using Milli-Q water. The template and primer were then combined with T4 ligase buffer (NEB) and diluted with water to the following final concentrations:

Component Concentration
10x T4 Ligase Buffer 1x
10 uM Template Stock 0.6 uM
10 uM Primer Stock 1.2 uM

Template Ligation

Following that, the sample was heated to 95C for five minutes and cooled to room temperature over the course of approximately 2.5 hours (a decrease in one degree every two minutes). Afterwards, 10 U of T4 ligase (NEB) was added for every microliter of the reaction. The sample was then subjected to one of the treatments listed above.

After the designated incubation time, the sample was heated to 65C for 10 minutes to deactivate the T4 ligase.

Phenolchloroform Extraction

Phenochloroform extraction was performed by first adding an amount of phenol:chloroform:isoamyl alcohol (25:24:1) (Thermo Fisher) equal to that of the sample and vortexing for 20 seconds. After centrifuging for five minutes at 16,000 g, the aqueous phase was removed and transferred to another tube. 

The following reagents were then added (glycogen was acquired from Thermo Fisher):

Component Concentration Amount
Glycogen 20 ug/uL 1 uL
Ammonium acetate 7.5 M 0.5x sample volume
Ethanol 100% 2.5x sample volume

The sample was then left to incubate at -20C for an hour, before being centrifuged at 4C for 30 min at 16,000 g. The supernatant was removed and the pellet was washed twice with 70% ethanol (resuspension followed by centrifugation at 4C for 2 min at 16,000g). Finally, the pellet was left to air dry for 10 min and resuspended in Milli-Q water.

Agarose Gel Electrophoresis

A 2% agarose gel was prepared using Top Vision Agarose (Thermo Fisher) and diluted 10x TBE buffer (Sigma-Aldrich). The samples were mixed with 6x purple loading dye (NEB) and ran at 120V until the dye front was two-thirds of the way down the gel. The gel was then imaged using [MACHINE NAME].


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. 


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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