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Materials & Methods

Materials

10×TBE Buffer

Content Volume/weight
Tris 108g
Boric acid 55g
Disodium Ethylenediamine 7.44g
Water Add to 1000ml
Total 1L

8M urea acrylamide solution (20%)

Content Volume/weight
Urea 192g
Acrylamide 77.3g
Bis-acrylamide 2.7g
10×TBE 40ml
Water Add to 400ml
Total 400ml

Denaturing polyacrylamide gels (8%)

Content Volume/weight
8M urea acrylamide solution(20%) 16ml
8M urea solution 24ml
APS 400μl
Total 40ml

Denaturing polyacrylamide gels(10%)

Content Volume/weight
8M urea acrylamide solution (20%) 20ml
8M urea solution 20ml
APS 400μl
TEMED 40μl
Total 40ml

Denaturing polyacrylamide gels (14%)

Content Volume/weight
8M urea acrylamide solution (20%) 28ml
8M urea solution 12ml
APS 400μl
TEMED 40μl
Total 40ml

Methods

The phosphorylation of ssDNA

Content Concentration
ssDNA 10μM
ATP 2mM
PNK 0.5U/μl
PNK buf.A

1. Mix the above reagents in a tube, heat the mixture in PCR Thermocycle Instrument to 37℃ and incubate for 15h;

2. heat up to 75℃ for 10 minutes to inactivate the kinase.

The strands to get cyclized need to be phosphorylated.

Preparation of single-stranded circular DNA

Content Concentration
phosphorylated ssDNA 0.6μM
splint 1.2μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

1. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure;

2. Anneal to 25℃ with the ramp of 0.1℃/s;

3. Add T4 DNA ligase and incubate in 25℃ for 3 hours;

4. Heat up to 65℃ for 10 minutes to inactivate the ligase.

Single-stranded DNA circle is an important material of our experiment.

ExonucleaseⅠto cleave non-circular ssDNA

Content Concentration
sample to digest \
EXO-I 0.95U/μl
EXO-I buf. 0.95×

1. Mix the above reagents in a tube, then put them in PCR Thermocycle Instrument, incubate in 37℃ for 2 hours;

2. Heat up to 75℃ for 15 minutes to inactivate the exonuclease I.

It’s used to get DNA circle purified, and also can distinguish the circular product from all kinds of bands in PAGE.

Phenol chloroform extraction

(Extraction agent A=Phenol: Chloroform: Isoamylol=25:24:1)

(Extraction agent B= Chloroform: Isoamylol=24:1)

1. Add the equal volume of agent A, and after vibration and centrifugation, discard the organic phase.

2. Add the equal volume of agent B, and after vibration and centrifugation, discard the organic phase.

Repeat these two steps twice.

Alcohol precipitation

1. Add 0.1× volume of pre-cooled 3M sodium acetate and 2.5× volume of pre-cooled absolute ethyl alcohol, and after vibration, cool it down to 25℃.

2. After 4℃ 12000rpm centrifugation for 10min, discard the supernatant and add 250μL pre-cooled 75% alcohol.

3. After 4℃ 12000rpm centrifugation for 10min, discard the supernatant and dry the precipitate.

4. Dissolve it in a certain volume of ddH2O.

By these two procedures we can get pure DNA circles and the first layer of our hauberk in high concentration.

Concentration of ssDNA is measured by Nanodrop. Agents are from Thermo. (3 for TTT hinge, 5 for TTTTT hinge, l for linear DNA, p for phosphorylated DNA, c for circular DNA.For example, C5p means phosphorylated strand B with TTTTT hinge, A3c means circle A with TTT hinge, and so on.)

1.Design of a two-ring catenane and exploration of the effect of using different hinges

AC3
Content Concentration
A3c 0.5μM
C3p 0.5μM
ACl3 1μM
ACr3 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AC5
Content Concentration
A5c 0.5μM
C5p 0.5μM
ACl5 1μM
ACr5 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

1. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure.

2. Anneal to 25℃ with the ramp of 0.1℃/s.

3. Add T4 DNA ligase and incubate in 25℃ for 3 hours.

4. Heat up to 65℃ for 10 minutes to inactivate the ligase.

(see results)

2.Exploring the topology of two-ring-catenane as the existence of staple and splint

CA5+
Content Concentration
C5c 0.5μM
A5p 0.5μM
spA 1μM
ACl5 1μM
ACr5 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AC5+
Content Concentration
A5c 0.5μM
C5p 0.5μM
spA 1μM
ACl5 1μM
ACr5 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AC52
Content Concentration
A5c 0.5μM
C5p 0.5μM
ACl5 1μM
ACr5 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AC51
Content Concentration
A5c 0.5μM
C5p 0.5μM
ACl5 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AC50
Content Concentration
A5c 0.5μM
C5p 0.5μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

1. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure.

2. Anneal to 25℃ with the ramp of 0.1℃/s.

3. Add T4 DNA ligase and incubate in 25℃ for 3 hours.

4. Heat up to 65℃ for 10 minutes to inactivate the ligase.

3.Exploring the effect of using scaffolds of different LD and hinge length

L1AB
Content Concentration
L1 0.25μM
A5p 1μM
B5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
L3AB
Content Concentration
L3 0.25μM
A5p 1μM
B5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
L5AB
Content Concentration
L5 0.25μM
A5p 1μM
B5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

1. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure.

2. Anneal to 10℃ with the ramp of 0.1℃/s.

3. Heat up to 25℃ with the ramp of 0.1℃/s;

4. Add T4 DNA ligase and incubate in 25℃ for 8 hours;

5. Heat up to 65℃ for 10 minutes to inactivate the ligase.

(see results)

4.Exploring the best procedure to produce four-ring catenane

4.1 Using the scaffold or an oligonucleotide as the splint

AB
Content Concentration
L3 0.5μM
A5p 1μM
B5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AsB
Content Concentration
L3 0.5μM
A5ps 1μM
B5p 1μM
spAs 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
ABs
Content Concentration
L3 0.5μM
A5p 1μM
B5ps 1μM
spBs 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AsBs
Content Concentration
L3 0.5μM
A5ps 1μM
B5ps 1μM
spAs 1μM
spBs 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

4.2 Using splints to control the topology

AsBs
Content Concentration
L3 0.5μM
A5ps 1μM
B5ps 1μM
spAs 1μM
spBs 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AB
Content Concentration
L3 0.5μM
A5p 1μM
B5p 1μM
spAs 1μM
spBs 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AB
Content Concentration
L3 0.5μM
A5p 1μM
B5p 1μM
spAs 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AB
Content Concentration
L3 0.5μM
A5p 1μM
B5p 1μM
spBs 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
AB
Content Concentration
L3 0.5μM
A5p 1μM
B5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

1. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure.

2. Anneal to 10℃ with the ramp of 0.1℃/s.

3. Heat up to 25℃ with the ramp of 0.1℃/s.

4. Add T4 DNA ligase and incubate in 25℃ for 8 hours.

5. Heat up to 65℃ for 10 minutes to inactivate the ligase.

(see results)

5.Exploring the best procedure to product eight-ring catenane

5.1 Using the bottom of the first layer as the splint

R2CD
Content Concentration
AB 0.5μM
C5p 1μM
D5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
R2CD
Content Concentration
AB 0.5μM
C5p 1μM
D5p 1μM
ACl5 2μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
R2C
Content Concentration
AB 0.5μM
C5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
R2C
Content Concentration
AB 0.5μM
C5p 1μM
ACl5 2μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
R2D
Content Concentration
AB 0.5μM
D5p 1μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

(The concentration of AB is calculated supposing its recovery is 100% in order to ensure that C5p and D5p is excess.)

1. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure.

2. Anneal to 10℃ with the ramp of 0.1℃/s.

3. Heat up to 25℃ with the ramp of 0.1℃/s.

4. Add T4 DNA ligase and incubate in 25℃ for 8 hours.

5. Heat up to 65℃ for 10 minutes to inactivate the ligase.

(see results)

5.2 Using an oligonucleotide as the splint

R2CsDs
Content Concentration
AB 0.5μM
C5ps 1μM
D5ps 1μM
spCs 2μM
spDs 2μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
R2Cs
Content Concentration
AB 0.5μM
C5ps 1μM
spCs 2μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
R2Ds
Content Concentration
AB 0.5μM
D5ps 1μM
spDs 2μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.
CsDs
(a negative control group and set to avoid disturbing of the polycatenane composed of circle C and D.)
Content Concentration
C5ps 1μM
D5ps 1μM
spCs 2μM
spDs 2μM
T4 DNA ligase 0.125 Weiss U/μl
T4 DNA ligase buf.

1. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure.

2. Anneal to 10℃ with the ramp of 0.1℃/s.

3. Heat up to 25℃ with the ramp of 0.1℃/s.

4. Add T4 DNA ligase and incubate in 25℃ for 8 hours.

5. Heat up to 65℃ for 10 minutes to inactivate the ligase.

(see results)