Experiment 

Material

We used DNA and biotin with the following structure in our experiments.

Experiment-1: Mixing dsDNA and avidin at different ratios

　In order to Determine the Optimal Avidin Concentration for the Formation of the Curved dsDNA-Avidin Complex,Electrophoresis was performed using a mixture of solutions with varying ratios of avidin- and biotin-modified dsDNA, and the distribution of bands was compared. 

Detailed experimental methods can be found in the appendix.

Experiment-2: Change the distance between biotin

　In order to confirm whether changes in the biotin-to-biotin distance affect the curvature of dsDNA, dsDNA with biotin-to-biotin distances of 1turn, 2turns, and 3turns was reacted with avidin, and electrophoresis was performed.

Detailed experimental methods can be found in the appendix.

Results and Discussion(Experiment₋1,2)

In Figures 5 to 8, we used a 10-bp DNA step ladder.

In Figure 9, we used a 50-bp DNA ladder.

〈Discussion〉

Comparing the lanes without and with avidin in Figures 5 and 6, the 20–40 bp bands are likely dsDNA, ssDNA, or short DNA fragments that do not have avidin attached.

For figure 9, there are two hypotheses regarding the bands appearing near the top. 

One hypothesis is that the band indicated by the arrow  indicates figure11 structure. Although avidin is in excess, after 1 turn steric hindrance between avidin molecules prevents the formation of the structure shown in the figure12, resulting instead in the structure. Therefore, in the two-turn and three-turn samples, where the distances between biotins are greater, the structure shown in the figure12 forms, and the corresponding bands disappear.

 The other hypothesis is that the band indicated by the arrow  indicates figure10 structure. In the figure10, there are no biotins that are unbound to avidin, so even with excess avidin, the bands remain in 1 turn. In 2 turn and 3turn, because the bending angle is steep, the structure in figure 10 is difficult to form. It is possible that avidin bound during the transition from the structure in figure 11 to that in figure 10, resulting in the structure shown in figure 12.  Therefore, in 2 turn and 3turn, the corresponding bands disappear.

We plan to mix DNA with a single biotin modification and avidin to form the structure shown in figure 11, and then compare the resulting band positions.

Experiment-3: Use the circularized DNA

　In order to confirm whether avidin-biotin binding can maintain the bent structure, cut the circular DNA bound to avidin with a restriction enzyme, and perform electrophoresis.

〈Methods〉

1, Circularize DNA-L-circle with circligase.

2, Remove non-circularized DNA with Exonuclease Ⅰ and Exonuclease Ⅲ.

3, Perform annealing with circularized DNA, DNA with biotins, and DNA-L-balance

4, Mix the circular dsDNA with avidin

5, Cut the circular dsDNA with EcoR1 at 37℃ for 60 minutes, then at 65℃ for 20minutes

6, Perform electrophoresis at 100V for 90 minutes at 4℃ 

Experiment-3-1

〈Purpose〉

Circularize DNA-L-circle with CircLigase

〈Methods〉

Added CircLigase to DNA-L-circle and incubated at 37 °C for 17 hours. 

Heated at 80 °C for 10 minutes to inactivate the enzyme. 

Heated the samples at 90 °C for 3 minutes to denature them, then performed electrophoresis at 100V for 70 minutes at room temperature.

〈Result〉

〈Discussion〉

Two bands were observed, one was circularized DNA and the other was non-circularized DNA.

It indicates successful circularization. 

Experiment-3-2

〈Purpose〉

Remove non-circularized DNA with Exonuclease Ⅰ and Exonuclease Ⅲ.

〈Methods〉

Added Exonuclease Ⅰ and Exonuclease Ⅲ to circularized DNA and incubated at 37 °C for 5 hours. 

Heated at 85 °C for 40 minutes to inactivate the enzyme. 

Used an Amicon 10K filter to isolate the circularized DNA. 

Heated the samples at 90 °C for 3 minutes to denature them, then performed electrophoresis at 100V for 70 minutes at room temperature.

〈Result〉

〈Discussion〉

The band corresponding to the linear (non-circularized) DNA disappeared, leaving only the band of the circularized DNA. 

This indicates that the removal of linear DNA was successful. 

Experiment-3-3

〈Purpose〉

The bands appearing in the circularized dsDNA after annealing are compared with the bands appearing in the circularized ssDNA and linear ssDNA before annealing.

〈Methods〉

Perform electrophoresis at 100V for 90 minutes at 4℃ 

〈Discussion〉

It was confirmed that some ssDNA remained after annealing, and the position of its band was identified.

Experiment-3-4

〈Purpose〉

Cut the circular DNA bound to avidin with a restriction enzyme, and perform electrophoresis.

〈Discussion〉

For all of the 1-, 2-, and 3-turn samples, there was no change in the band pattern between the samples with and without avidin when the restriction enzyme was added. 

It is possible that the avidin–biotin interaction cannot maintain the bent structure. 

But, avidin may degrade under high-temperature conditions in the presence of EDTA. 

I used TE, which include EDTA, as the reaction buffer for binding avidin and biotin. And heated to inactivate EcoR1.

Using a buffer without EDTA might allow the avidin-bound structure to remain bent.