Appendix

Simulation

２, Other combinations

Biotin spacing：4.6nm(1 turn + major groove)

Biotin spacing：5.6nm(1 turn + minor groove)

３, Effect of Biotin Spacing on Curvature

Using Pymunk's rigid body approximation model, we investigated how the curvature of the bent structure differs for biotin inter-distance values of 4.6nm(1 turn + major groove) and 5.6nm(1 turn + minor groove).

The length of the biotin linker was fixed at 3.05 nm, the value actually used in the experiment.

The curvature radius is measured at the point when the DNA's curved structure has finally stabilized. However, if the curved structure collapses, the value is taken immediately before the collapse occurs.

Source code

Experiment

〇Annealing

An annealing solution was prepared with the composition shown in Table 1.Perform annealing at 95°C for 3 minutes, then cool 　at -1°C/minute to 4°C.

Experiment 1: Mixing dsDNA and avidin at different ratios

Experiment-2: Change the distance between biotin

<Objective>

Experiment1

To determine the Optimal Avidin Concentration for the Formation of the Curved dsDNA-Avidin Complex.

Experiment2
To confirm whether changes in the biotin-to-biotin distance affect the curvature of dsDNA

<Methods＞

Experiment-1 and Experiment-2 were conducted simultaneously.

1,Avidin solution prepareration

   We mixed 2 μL of avidin with 333 μL of MQ by pipetting.2 μL was taken, mixed with 8 μL of MQ, and the concentration was measured three times using DeNovix(Table 2).Then,the 12.25 μM solution was diluted to 5 μM and dispense into 60 μL.

2,React biotin-modified dsDNA with various avidin concentrations

　Prepare a mixture of avidin and biotin-modified dsDNA at the ratios specified in Table 3, varying the avidin concentration.　Experiments were conducted using avidin-to-biotin-modified dsDNA ratios of 0.25:1, 0.5:1, 1:1, 4:1, 6:1, 8:1, and 10:1. Additionally, samples without added avidin in solution were prepared.For each ratio, experiments were conducted using four patterns: biotin spacer distance of 1 turn, 2 turns, 3 turns, and no biotin modification(1turn=3.4nm).

3,Ladder and sample preparation

   Prepare a ladder, and sample solutions in the following proportions.

4,Preparation of gels for electrophoresis

   Degassed for 5 minutes before adding 10% APS.

5,Electrophoresis

   Stained with SYBR Green II for 15 minutes, washed in 1x TBE buffer for 1 minute, and then photographed.

Experiment1,2-1:Electrophoresis with no avidin

<Objective>

To check the band position in the absence of avidin. 

<Methods>

Make 15% Native PAGE

Make a ladder and samples

<Result>

<Discussion>

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

Experiment1,2-2:Compare ssDNA and dsDNA

<Objective>

To check the band position when ssDNA was mixed with avidin.

<Methods>

Make 15% Native PAGE

Make avidin-biotin solution(dsDNA 0.25:1 0.5:1 1:1 2:1, ssDNA 1:1)

Make a ladder and samples

Perform electrophoresis at 150V for 80 minutes at 4℃

<Result>

<Discussion>

The band position corresponding to the ssDNA–avidin complex was identified. 

1.Annealing solution preparation

   An annealing solution was prepared with the composition shown in Table below.Perform annealing at 95°C for 3 minutes, then cool at -1°C/minute to 4°C.

2.Preparation of avidin-biotin reaction mixture

3.Incubate at 60°C for 17 hours and at 80°C for 10 minutes

4.Preparation of gels for electrophoresis

   The gels were degassed for 5 minutes before adding 10% APS.

5.Preparation of sample and ladder

6.Denatured at 95°C for 3 minutes, electrophoresed at 100V for 70 minutes at room temperature, stained with SYBR Green for 15 minutes, and washed with 1x TBE.

7.Remove non-circularized DNA

   To 99μL of circligase reaction mixture, 4.48μL of Exo I and 4.48μL of Exo III were added on ice. 

Incubated at 37°C for 5 hours, 85°C for 40 minutes, and cooled to 25°C at -1°C per minute. 

392μL of MQ was added to make a 500μL volume, and the mixture was placed in an Amicon 10K. 

The mixture was centrifuged at 14,000G for 10 minutes at room temperature. 

Another 500μL of MQ was added, and the mixture was centrifuged at 14,000G for 10 minutes at room temperature. 

The mixture was then turned upside down and centrifuged at 1,000G for 2 minutes at room temperature. 

The DNA attached to the filter was removed.

8.The concentration of the 4-fold diluted solution was measured in triplicate using DeNovix.

9.Preparation of gels for electrophoresis

   The gels were degassed for 5 minutes before adding 10% APS. The same gel were used before.

10.Preparation of sample and ladder

11.Electrophoresis

Denatured at 95°C for 3 minutes, electrophoresed at 150V for 60 minutes at room temperature, stained with SYBR Green, and washed with 1x TBE.

Experiment 3-3:After attaching avidin to the circularized DNA, it is cut with a restriction enzyme.

〈Purpose〉

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

1.Preparation of gels for electrophoresis

   The solution was degassed for 5 minutes before adding 10% APS.

2.Annealing solution preparation

   95°C for 3 minutes, cooled at -1°C/min

3.Avidin-biotin reaction mixture preparation

   Incubated at room temperature for 30 minutes.

4.Biotin solutions preparation

5.Mix the avidin-biotin reaction mixture with the biotin solution

   Incubated for 30 minutes.

6.EcoR1 reaction mixture preparation

   37°C for 1 hour, then 65°C for 20 minutes

7.Preparation of sample (finished product, after annealing, after mixing with biotin solution, EcoR1 treatment without avidin)

<Result>

See Experiment3-3 (Experiment page)

1.Preparation of gels for electrophoresis

   The solution was degassed for 5 minutes before adding 10% APS.

2. Preparation of ladder and sample(circularized dsDNA, circularized ssDNA, ssDNA without biotin, balance)

3.Electrophoresis

   Electrophoresis at 4°C for 90 minutes at 100V

Stain with SYBR Green II for 15 minutes

Wash with 1x TBE for 2 minutes