Biomod/2013/Sendai/protocol

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Protocol


Step 2 Chain-reactive burst

i)Bending approach

1)Making DNA origami

1-1)Making DNA origami

DNA origami recipe

We designed DNA origami by <A Href="http://cadnano.org/">caDNAno2</A>, 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 DNAs complementary to the unique sequence are expected to easily hybridize with the unique sequence.
The 30nt single-stranded parts are stable till 37 degrees, according to <A Href="http://www.nupack.org/">NUPACK</A>).
The 141 staples have the same length so that they may place 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 DNAs.
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 ccDNA) 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 cc DNA and fluorescent tagged DNA with the same unique single-stranded parts of our origami, we arranged two kinds of adaptor DNAs (at the third and fourth in Table1). One adaptor has complementary sequences to both the unique sequence and cc 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 DNA strands Its sequence
Cholesterol-conjugated DNA (ccDNA) CCAGAAGACG
Fluorescent tagged DNA ACTAGTGAGTGCAGCAGTCGTACCA
Adaptor strand for cc DNA and the unique sequence in DNA origami CGTCTTCTGGCTCTCGATGCGACAG
Adaptor strand for fluorescent tagged DNA and the unique sequence in DNA origami TGGTACGACTGCTGCACTCACTAGTCTCTCGATGCGACAG

Table.1 The sequence of the strands used in our experiment

Annealing

The 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).

<ur>
  • Annealing solution with fluorescent tagged DNAs 50µl
    84nM M13mp18 2.38µl
    Staples
    1µM migihaji 1µl
    1µM hidarihaji 1µl
    1µM ashibatemae 1µl
    200nM ashiba 5µl
    1µM cholesterol-hybridizing ssDNA 3µl
    1µM fluorescent-tagged DNA-hybridizing ssDNA 3µl
    5xTAE Mg2+ 10µl
    mQ 20.62µl
    1µM fluorescent-tagged DNA 3µM

    • Table.2 Annealing solution with fluorescent tagged DNAs

  • Annealing solution with no fluorescent tagged DNAs (control) 50µl
    We changed 3µl fluorescent tagged DNAs in the above solution into the same quantity of mQ.


    • AFM observation

    As we thought excess staples produced more aggregation and made AFM observation difficult, control annealing solution was used for AFM observation.

    1-2)Labeling DNA origami

    We confirmed that our DNA origami was fluorescently labeled by electrophoresis.

    50µl of Annealing solution with fluorescent tagged DNAs (used in 1-1)Making DNA origami) contains 3µl of 1µM fluorescent tagged DNAs.
    To see if the origami binds to the fluorescent tagged DNA in shorter time, we added 0.6µl of 1µM fluorescent tagged DNAs 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.

    2)Destroying liposomes

    2-1) Making liposomes

    We made liposome that was to be broken by DNA origami.
    First we mixed 1µl lipid (10mM DOPC) and 99µl solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then we left it for one night in a vacuum dryer. After drying, we added 100µl of the same buffer as that of DNA origami (1xTAE Mg2+) into the sample and heat it in warm water (about 90 degrees) for a few hours.

    2-2) Investigating the interaction of DNA origami and liposomes

    To float cc DNAs on the surface of liposome, we added cc DNAs into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Each sample was as follows.

    <ur>
  • Liposome with 0.018µM cc DNAs: 1µl 0.1µM cc DNAs and 2.5µl liposome
  • Liposome with 0.069µM cc DNAs: 10µl 0.1µM DNAs and 2.5µl liposome
  • Liposome with 1.8µM cc DNAs: 1µl 10µM DNAs and 2.5µl liposome
  • Liposome with 6.9µM cc DNAs: 10µl 10µM DNAs and 2.5µl liposome

    • We 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.

    84nM M13mp18 2.38µl
    Staples
    1µM migihaji 1µl
    1µM hidarihaji 1µl
    1µM ashibatemae 1µl
    200nM ashiba 5µl
    100µM cholesterol-hybridizing ssDNA 4.23µl
    100µM fluorescent-tagged DNA-hybridizing ssDNA 4.23µl
    5xTAE Mg2+ 10µl
    mQ 23.54µl

    Table.3 50µl Annealing solution for fluorescent microscope observation

    After annealing, we added 4.23µl 100µM fluorescent-tagged DNA (the same quantity of fluorescent-tagged DNA-hybridizing ssDNA).

    2-3)Counting liposomes

    For the sake of observation convenience, we mixed 1µl 1µM TR-DHPE (red fluorescent dye) with 1µl lipid (10mM DOPC) and 98µl solvate (CHCl3) in a microtube, and desiccate it with Argon gas. Then we left it for one night in a vacuum dryer. After drying, we added 100µl 1xTAE Mg2+ into the sample and heat it in warm water (about 90 degrees) for a few hours.

    After liposome was made, we added 1µl 10µM cc DNA into 2.5µl liposome (the final concentration of cc DNA was 1.8 µl). We counted the number of liposomes with a fluorescent microscope.
    After counting, we added 2µl DNA origami and counted the number of liposomes again. For control, we changed 2µl DNA origami into 2µl 1xTAE Mg2+ buffer.


    ii)Flower micelle approach

    2)Confirming the hybridization of trigger and loop DNA

    We checked whether trigger DNA hybridizes with loop DNA at normal temperature by electrophoresis.

    30% Acryl amide 3.3ml
    mQ 5.59ml
    10xTAE 1ml
    TEMED 10µl
    10xAPS 100µl

    Table.4 10% Acrylic amide gel recipe

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