1 Step1 温度感受性リポソームの破壊
Structure of NIPAM
|Egg York PC(10mM)||10µl|
1. Drying the liposomes above with argon gas and letting them stand for a night
2. Adding L paraffin 100µl to 1 and sonicating them for an hour
3. Picking up 10µl from 2, adding 25μl NIPAM2mg/ml to them and vibrating them with Vortex
2 Step2 DNAによる連鎖的リポソームの破壊
Making DNA origami
DNA origami recipe
We designed DNA origami by caDNAno2, software for designing 2D and 3D DNA origami.
Our DNA origami has 141 staples that have 30nt free single-stranded parts outside the DNA origami. The sequence of the parts is “each DNA origami staple-TTTTTTTTTTTTTTTCTGTCGCATCGAGAG”.
Between the staple and unique (CTGTCGCATCGAGAG) sequences, 15 T bases are inserted. They are to make a T loop. Thanks to this T loop, single-stranded DNA complementary to the unique sequences (such as Anchored DNA) are expected to easily hybridize with the unique sequence.
The 30nt single-stranded parts are stable till 37 degrees, according to NUPACK).
The 141 staples have the same length so that they may be present at the same intervals in the DNA origami.
Each side of our origami is not fully covered with staples, and single-stranded M13 remains. This is for preventing π-π interaction and stacking by hydrophobic interaction between base pairs of double-stranded DNA.
This design enables each DNA origami to exist individually.
The list of strands
The other strands exept DNA origami staples used in our experiment are shown in Table1.
The sequence of cholesterol-conjugated DNA (in the rest of this document, referred to as Anchored DNA) is shown below (at the first sequence in Table1). For labeling, we also attached fluorescent tagged DNA (at the second in Table1) to our DNA origami.
To hybridize different strands of Anchored DNA and fluorescent tagged DNA with the same unique single-stranded parts of our origami, we arranged two kinds of adaptor DNA (at the third and fourth in Table1). One adaptor has complementary sequences to both the unique sequence and Anchored DNA. The other has complementary sequences to both the unique sequence and the fluorescent tagged DNA. Thanks to these two adaptors, two different strands can bind to the same unique sequence.
|The kinds of DNAtrands||Its sequence|
|Cholesterol-conjugated DNA (Anchored DNA)||CCAGAAGACG|
|Fluorescent tagged DNA||ACTAGTGAGTGCAGCAGTCGTACCA|
|Adaptor strand for Anchored DNA and the unique sequence in DNA origami||CGTCTTCTGGCTCTCGATGCGACAG|
|Adaptor strand for fluorescent tagged DNA and the unique sequence in DNA origami||TGGTACGACTGCTGCACTCACTAGTCTCTCGATGCGACAG|
Annealing of DNA origamiThe annealing solution is shown in Table2. The annealing was conducted for 2 hours and 51minutes (from 95 to 25 degrees: lower 1 degree per 2 minutes).
|1µM cholesterol-hybridizing ssDNA||3µl|
|1µM fluorescent-tagged DNA-hybridizing ssDNA||3µl|
|1µM fluorescent-tagged DNA||3µM|
We changed 3µl fluorescent tagged DNA in the above solution into the same quantity of mQ.
AFM observationAs we thought excess staples produced more aggregation and made AFM observation difficult, control annealing solution was used for AFM observation.
ElectrophoresisWe confirmed that our DNA origami was fluorescently labeled by electrophoresis.
50µl of Annealing solution with fluorescent tagged DNA (used in 1-1)Making DNA origami) contains 3µl of 1µM fluorescent tagged DNA.
To see if the origami binds to the fluorescent tagged DNA in shorter time, we added 0.6µl of 1µM fluorescent tagged DNA into 10 µl control annealing solution, and left it for 40 minutes.
Agarose gel recipe: 0.4g agarose, 0.8ml 50xTAE, 39.2ml mQ
The electrophoresis was conducted with 1% agarose gel, CV 100V, for 50 minutes.
Concentration of Anchored DNATo float Anchored DNA on the surface of liposome, we added Anchored DNA into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Each sample was as follows.
Observation by phase and fluorescent microscopeWe observed each sample with a phase microscope.
Then we added 2µl DNA origami into each sample and saw if some change would happen with a fluorescent microscope.
The DNA origami for fluorescent microscope observation was made according to Table3 annealing solution. It contained more cholesterol-hybridizing ssDNAs and fluorescent-tagged DNA-hybridizing ssDNAs than Annealing solution used in 1-1), because we considered a sample with more fluorescent molecules was suitable for observation.
|100µM cholesterol-hybridizing ssDNA||4.23µl|
|100µM fluorescent-tagged DNA-hybridizing ssDNA||4.23µl|
After annealing, we added 4.23µl 100µM fluorescent-tagged DNA (the same quantity of fluorescent-tagged DNA-hybridizing ssDNA).
Making liposomeWe made liposomes in a spontaneous-transfer way. They were divided into two types: liposomes A of GFP, Green Fluorescent Protein, and liposomes B of Red Fluorescent Protein. These two kinds of liposomes have the same Outer Buffer but different Inner Buffer. Composition of these two buffers is as follows.
|LiposomeA Inner Buffer||GFP||5µl|
|LiposomeB Inner Buffer||ローダミン||0.5µl|
2. Putting paraffin 50 on outer 50
3. Putting 1 on 2
4. Centrifuging 3 for 5 minutes
5. Observing leak of liposomes from the bottom of tubes by needles