MATERIALS&METHODS

Materials&Methods

Materials

10×TBE Buffer

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

NEBuffer 1.1

Content Concentration
Bis Tris Propane-HCl 10mM
MgCl2 10mM
BSA 100μg/ml
pH7.0@25℃
 

Acrylamide solution(30%)

Content Volume/weight
Acrylamide 290g
Bis-acrylamide 10g
Water Add to 1000ml
Total 1l
 

8M urea acrylamide solution (20%)

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

Denaturing polyacrylamide gels(8%)

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

Steps: Dissolve and gently mix the above reagents in a beaker of 200 ml, then pour the gel into a tank. Carefully insert comb into gel sandwich until bottom of teeth reaches top of front plate. After the gel is solidified, samples are loaded. Run the gel by 350V for about 3 hours.

Native polyacrylamide gels(8%)

Content Volume
Acrylamide solution(30%) 11mL
100mM MgCl2 4mL
10×TBE 10mL
water 21ml
TEMED 40μL
Ammonium persulfate(10%) 400μL
 

Steps: Dissolve and gently mix the above reagents in a beaker of 200 ml, then pour the gel into a tank. Carefully insert comb into gel sandwich until bottom of teeth reaches top of front plate. After the gel is solidified, samples are loaded. Run the gel by 250V with ice to reduce the temperature for about 3 hours.

Denaturing polyacrylamide gels with urea and methanamide(14%)

Content Volume
Acrylamide solution(45%) 31.15mL
10×TBE 10mL
Methanamide 24mL
Urea 42g
TEMED 40μL
Ammonium persulfate(10%) 400μL
 

Steps: Dissolve and gently mix the above reagents in a beaker of 200 ml, then pour the gel into a tank. Carefully insert comb into gel sandwich until bottom of teeth reaches top of front plate. After the gel is solidified, samples are loaded. Run the gel by 350V for about 3 hours.

Methods

Preparation of Z-B chimera

1. Preparation of ssDNA circle:

1) Phosphorylation of single strand DNA in 5’ end.

All DNA strands were synthesized and purified by GENEWIZ Bio-Technique Co. Ltd. We need 5μl of original material(single-stranded DNA)to make the final concentration be 5μmol/L, 2μl of ATP to provide phosphate group, 2μl of PNK ligase to promote phosphorylation, 2μl of PNK buffer A to provide other ion conditions, and 10μl of ddH2O to add the volume to 20μl. Mix the above reagents in a tube, heat the mixture in PCR Thermocycle Instrument to 37℃ and incubate for a whole night, then heat up to 75℃ for 10 minutes to inactivate the ligase.

2) Circularization of single strand DNA by using splint.

We need 1.6μl of single strand DNA solution after phosphorylation from the step 1 to make the final concentration be 0.1μmol/L, 0.8μl splint to promote the circularization of single strand DNA, 8μl T4 DNA ligase buffer, 2μl T4 DNA ligase, and 67.6μl ddH2O to add the volume to 80μl. Mix the above reagents and heat the mixture to 90℃ for 3 minutes to open the secondary structure, then incubate in 25℃ for 3 hours, finally, heat up to 65℃ for 10 minutes to inactivate the ligase.

3) ExonucleaseⅠto cleave non-circular single strand DNA.

Keep the proportion of sample, exonucleaseⅠand exonucleaseⅠin 9:1:0.5. Mix the above reagents in a tube, then put them in PCR Thermocycle Instrument, react in 37℃ for 2 hours, then react in 75℃ for 15 minutes to inactivate the exonucleaseⅠ.

4) Phenol chloroform extraction and alcohol precipitation.

Phenol chloroform extraction is to wipe off protein, polysaccharide and phenolic substance and alcohol precipitation is to dissociate the nucleic acid from the system.

2. Preparation of Z-B DNA chimera:

1) Base pairing reaction between two circular DNAs with complementary sequences.

Mix the products from the step 1 4)(linear α and its complementary linear β)up in another tube and keep their proportion in 1:1, add 10×NEB1.1 buffer and ddH2O to the tube, react in 90℃ for 3 minutes to open the secondary structure, then ramp down to 25℃ by 0.1℃/s and react for 10 minutes.

2) Preparation of reference samples.

(1) Base pairing reaction between circular DNA and its complementary ssDNA.

Mix up the circular DNAs and its complementary ssDNA and keep their proportion in 1:1, add 10×NEB1.1 buffer and ddH2O to the tube, react in 90℃ for 3 minutes to open the secondary structure, then ramp down to 25℃ by 0.1℃/s and react for 10 minutes.

(2) Base pairing reaction between complementary linear DNAs.

Mix linear α and it’s complementary linear β in 1×NEB1.1 reaction buffer and keep their proportion in 1:1, add ddH2O to the tube, heat up to 90℃ for 3 minutes to open the secondary structure, then cool down to 25℃ ( 0.1℃/s) and keep for 10 minutes.

(3) Reaction between ss-cirDNA and it’s complementary linear DNA withT4 DNA ligase.

Mix circle DNA and it’s complementary linear DNA in 1×T4 DNA ligase reaction buffer ( 4.5μl ) and keep their proportion in 1:1, add 0.5μl T4 DNA ligase and ddH2O to the tube, heat up to 80℃ for 3 minutes to open the secondary structure, then cool down to 25℃ ( 0.1℃/s )and keep for 2 hours, inactivate enzyme by heating the mixture up to 65℃for 10 minutes. Then the closed B-DNA circles were prepared.

3. Gel electrophoresis:

Z-B DNA chimera was analyzed on an 8% native polyacrylamide gel with 10mM MgCl2 under 250V and an 8% denaturing polyacrylamide gel under 350V respectively. The native polyacrylamide gels were run in 1X TBE buffer with 1mM MgCl2 at the temperature under 20℃ and the denaturing polyacrylamide were run in 1×TBE at ambient temperature. ( The preparation of 8% native polyacrylamide gel and the condition of running buffer were optimized, the reason why we optimized the experiment condition will debate in Discussion 1.) When other lengths of Z-B chimera were analyzed, the native polyacrylamide gels were run in 1X TBE buffer at the temperature under 20℃ and the denaturing polyacrylamide were run in 1×TBE at ambient temperature. Samples were prepared as below:

The preparation of ladder

Content Volume
Ladder* 0.5μL
6x loading buffer 2μL
Total 2.5μL
 

*Ladder: GeneRuler Low Range DNA ladder ready-to use #SM1203 (Thermo scientific) DNA bands were visualized by SYBR Green II staining (Life technologies) and detected by Gel Doc™ XR+ System (Bio-Rad). Bands were analyzed by Image Lab 3.0 software (Bio-Rad).

The preparation of samples

Content Volume
ssDNA(5μM) Sample 0.2μL
6x loading buffer 2μL
Total 2.2μL
Circular DNA(1μM) Sample 1μL
6x loading buffer 2μL
Total 3μL
Z-B DNA chimera(0.25μM) Sample 4μL
6x loading buffer 2μL
Total 6μL
B-DNA(0.25μM) Sample 4μL
6x loading buffer 2μL
Total 6μL
DNA duplex(0.25μM) Sample 4μL
6x loading buffer 2μL
Total 6μL
 

The transcription of Z-B chimera

The first two steps are the same as the preparation of Z-B chimera:

1. Preparation of ssDNA circle

2. Preparation of Z-B DNA chimera

3. Preparation of transcription product of DNA duplex:

We built up a system including 0.2μg linear α and linear β, 2μl 5×transcription buffer, 1μl 5M NTPs, 0.5μl RNase inhibitor, 0.5μl T7 promoter and added 3.18μl ddH2O to add the volume to 10μl. Incubate in 25℃ for 2 hours and heat the mixture up to 70℃for 10 minutes. After reaction, extract 5μl of the reaction solution and add 0.2μl DNaseⅠ, then react in 37℃for 15 minutes and heat up to 65℃for 10 minutes after adding 0.2μl 0.5M EDTA to prove the product left is RNA.

4. Preparation of transcription product of ss-cirDNA:

We built up a system including 0.2μM circle α or circle β, 2μl 5×transcription buffer, 1μl 5M NTPs, 0.5μl RNase inhibitor, 0.5μl T7 promoter and added 4μl ddH2O to add the volume to 10μl. Incubate in 37℃for 2 hours and heat the mixture up to 70℃for 10 minutes.

5. Preparation of transcription product of Z-B DNA chimera:

We built up a system including 0.2μM circle α and circle β, 2μl 5×transcription buffer, 1μl 5M NTPs, 0.5μl RNase inhibitor, 0.5μl T7 promoter and added 2μl ddH2O to add the volume to 10μl. Incubate in 37℃for 2 hours and heat the mixture up to 70℃for 10 minutes.

6. Preparation of transcription product of closed B-DNA circles:

First, we built up a system including 0.5μM circle α and circle β, 0.5μl T4 buffer, 0.5μl T4 DNA ligase and added 1μl ddH2O to add the volume to 5μl, react in 80℃ for 3 minutes to open the secondary structure, ramp down to 30℃by 0.1℃/s and react for 2 hours, and heat the mixture up to 65℃for 10 minutes to prepare the closed B-DNA circles. Then, we built up a system including 0.2μM closed B-DNA circles, 2μl 5×transcription buffer, 1μl 5M NTPs, 0.5μl RNase inhibitor, 0.5μl T7 promoter and added 2μl ddH2O to add the volume to 10μl. Incubate in 37℃for 2 hours and heat the mixture up to 70℃for 10 minutes.

7. Gel electrophoresis:

The transcription product of Z-B DNA chimera was analyzed on an 12% denaturing polyacrylamide gel under 350V respectively. The denaturing polyacrylamide were run in 1×TBE at ambient temperature.

Preparation of Z-B RNA chimera

1. Preparation of ssRNA circle:

1) Circularization of single strand RNA by using T4 RNA ligase directly.

All RNA strands were synthesized, phosphorylated and purified by Genscript biotechnology. We need 10μl of single strand RNA solution to make the final concentration be 10μmol/L, 0.5μl RNase inhibitor to inhibit the degradation of RNA, 10mM ATP, 50%PEG 8000, 2μl T4 RNA ligase buffer, 1μl T4 RNA ligase, and 2.4μl ddH2O to add the volume to 20μl. Mix the above reagents and incubate in 25℃ for 2 hours, then, heat up to 65℃ for 15 minutes to inactivate the ligase.

2) Circularization of single strand RNA by using DNA splint.

We need 1μl of single strand RNA solution to make the final concentration be 0.5μmol/L, 1μl DNA splint to promote the circularization of single strand RNA, 0.5μl RNase inhibitor to inhibit the degradation of RNA, 2μl T4 DNA ligase buffer, 0.5μl T4 DNA ligase, and 15μl ddH2O to add the volume to 20μl. Mix the above reagents and heat the mixture to 75℃ for 3 minutes to open the secondary structure, then incubate in 25℃ for around 3 hours, finally, heat up to 65℃ for 10 minutes to inactivate the ligase.

2. Preparation of Z-B RNA chimera:

Mix the products from the step 1(circle α and its complementary circle β)up in another tube directly and keep their proportion in 1:1, react in 75℃ for 3 minutes to open the secondary structure, then ramp down to 25℃ by 0.1℃/s and react for 20 minutes.