Biomod/2013/Sendai/experiment

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J. Am. Chem. Soc., 2004, 126 (8), pp 2290–229 Publication Date (Web): February 3, 2004<br><br>
J. Am. Chem. Soc., 2004, 126 (8), pp 2290–229 Publication Date (Web): February 3, 2004<br><br>
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<h5>Result</h5>
<h5>Result</h5>

Revision as of 06:07, 27 September 2013

Biomod2013 Sendai ver2.0

Biomod2013
  Team
Sendai

Experiment

Fig.11 AFM image of DNA origami made by urea diluting annealing

Discussion
We observed DNA origami we designed. However, we also observed a lot of sheet structures like fragments. We suppose that some staples did not hybridize with M13 DNA caused these fragments. Rapidly dilution of urea may cause the low yield of objective DNA origami, because stirring make urea removing faster. Nevertheless, it is necessary to measure the yield of DNA origami under low speed dilution. We should note that, in the egg-type initiator, the speed of diluting urea would be later than the speed of this experiment, and thus fast dialysis is not essential to form DNA origami in our system.
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Step 2 Chain-reactive burst

Once the trigger DNA, which begins the interaction, is released, the next is the chain-reactive burst. If a liposome containing new triggers and active ingredients is disrupted, the released triggers come to collapse the surrounding liposomes one after another.
We tackled the problem of destroying liposomes by the following two approaches.
  • i)Bending approach
  • ii)Flower micelle approach

  • i)Bending approach

    Experiment list

    The experiment necessary for realization of Bending approach is following.
    1)Making DNA origami
    1-1)AFM observation
    1-2)Labeling DNA origami
    2)Collapsing liposomes
    2-1)Making liposomes
    2-2)Investigating the interaction of DNA origami and liposomes
    2-3)Counting liposomes


    1)Making DNA origami

    1-1)AFM observation

    Purpose
    In our project, we used DNA origami as triggers for collapsing liposomes. We designed a rectangular DNA origami with a chipped edge and tried to make it.

    Principle
    DNA origami is a method applied to making nano-structures of various shapes. DNA origami consists of two kinds of strands: scaffold and staples. Scaffold is a long round single-stranded DNA, and staples are short linear single-stranded DNAs. By annealing scaffold and designed staples, we can easily get DNA origami of our own design.

    Method
    We mixed M13mp18, staples, 5xTAE Mg2+, and mQ in a microtube and annealed it for 2.5 hours.
    Protocol

    Result
    We confirmed that our DNA origami was well formed by AFM (Atomic Force Microscope) (Fig.1).

    Fig.1 AFM image of DNA origami (M13: 4nM, staples:20nM)

    Discussion
    Just like our design, rectanglar origamis with chipped edges were observed.


    1-2)Labeling DNA origami

    Purpose
    If the origami is fluorescently labeled, it is much easier to observe the effect of DNA origami on liposomes. So we labeled our origami by hybridizing it with fluorescent tagged DNA strands.

    Method
    Our DNA origami has many staples that can bind to fluorescent tagged DNAs for labeling. We mixed fluorescent tagged DNAs together with DNA origami staples in annealing solution.
    In addition, to see if the origami binds to the fluorescent tagged DNA in shorter time, we added the fluorescent tagged DNA into control annealing solution, which contained no fluorescent tagged DNA, and left it for 40 minutes.
    To see the origami was well labeled with fluorescent molecules, we used electrophoresis.
    Electrophoresis was conducted with a 1% agarose gel, CV100V for 50 minutes.
    Protocol

    By scanning a gel before staining, we can see only the bands of DNA structures with fluorescent molecules; scanning a gel after staining, we can see the bands of all DNA structures. So we scanned a gel before and after staining (we scanned both a non-stained and a stained gel).
    First we saw the bands of our origami in a non-stained gel. Then, we compared the bands with those in a stained gel. If the bands of origami in a non-stained gel were at the same height as those in a stained gel, we can say that our origami is successfully fluorescently labeled.

    Result
    In a non-stained gel (Fig.2), only bands in lane 3 and 4 from the left (*Ori, **Ori) can be seen. They are fluorescent labeled structures. In addition, as they gave the same result, 40 minutes is long enough for fluorescent labeling.

    Fig.2 Non-stained gel image: only bands in two lanes can be seen. From the left, they are DNA origami with fluorescent molecules in pre-annealing (Ori*), and DNA origami with fluorescent molecules in post-annealing (Ori**)

    In a stained gel (Fig.3), marker (lane 1) had the longest DNA strand of 20kb. Comparing this and M13mp18 (lane 2) with annealed DNA origamis (lane 3,4,5), the bands of the origamis are at the higher position. Therefore, we concluded that in lane3~5, DNA origami structure made of M13 and staples were made as we had expected.
    We considered that the bands in lane3~5 are seen as if they were diffused, just because our origami has many staples binding to the fluorescent tagged DNAs, and each origami attaches to different number of them, and its molecular weight varies.

    Fig.3 Stained gel image: from the left, marker, M13mp18, Ori*, Ori**, and DNA origami with no fluorescent molecule (Ori)

    Discussion
    Combining the results of Fig.2 and 3, the fluorescent labeled bands in lane3 and 4 in Fig.2 are at the same height as those of DNA origami in Fig.3. Thus, we concluded our origami was successfully fluorescently labeled.

    2)Collapsing liposomes

    2-1) Making liposomes

    Purpose
    We make liposomes that are to be collapsed by DNA origami.

    Principle
    Phospholipids, which compose liposomes, are amphipathic molecules. They have hydrophilic and hydrophobic groups, and when they touch water, they make micelles: some hydrophilic groups take water inside. At the same time, other hydrophilic groups touch the water outside. So they form the innermost and outermost part of a micelle. On the other hand, the hydrophobic groups form the intermediate part of a micelle.
    In this way, spherical liposomes are made.

    Method
    To make liposomes, first we mixed lipid (DOPC) and solvent (CHCl3) in a microtube, and desiccate it with Argon gas. Then, adding some buffer (1xTAE Mg2+), we heated it in warm water for a few hours.
    Protocol

    The result and discussion are integrated in the next passage of (2-2) Investigating the interaction of DNA origami and liposomes.


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

    Purpose
    To collapse liposome with our origami, first we investigated how our DNA origami affected liposomes.

    Principle
    To collapse liposomes with our origami, many origamis have to hybridize with the surface of liposomes.
    To begin with, we added cholesterol-conjugated single-stranded DNAs (in the rest of this document, referred to as aptamer) into liposomes, and made them float on the surface. If the aptamer have a complementary part to our origami, the origami is expected to hybridize with the surface. In this way, many origamis would hybridize with liposome via aptamers.

    Method
    We added aptamers into liposomes at the final concentration of 0.018, 0.069, 1.8, and 6.9µM. Then we observed the samples with a phase microscope. Next, adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.
    Protocol

    Result
    In all four conditions, liposomes were observed with a phase microscope. We confirmed the formation of multilamella liposomes (Fig.4~7).


    Fig.4 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)


    Fig.5 Phase microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)


    Fig.6 Phase microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)


    Fig.7 Phase microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)

    Adding fluorescently labeled DNA origamis into the above liposomes, we saw if some change would happen with a fluorescent microscope.
    When the concentration of aptamer was 0.018, 0.069µM, many gleaming (in green color) liposomes were observed. We confirmed that the fluorescently labeled origamis well hybridized with the liposomal surface (Fig.8,9,10).
    Fig.8,9 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.018µM)

    Fig.10 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 0.069µM)

    On the other hand, when the concentration of aptamer was 1.8µM, few gleaming liposomes could be seen with a fluorescent microscope (Fig.11). This result indicates the possibility that liposomes were collapsed.

    Fig.11 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 1.8µM)

    When the concentration of aptamer is 6.9µM, some liposomes were gleaming and others distorted, forming networks (Fig.12).

    Fig.12 fluorescent microscope image of liposomes (cholesterol-conjugated DNA: 6.9µM)

    Discussion
    From these results, we put forward the following hypothesis about the interaction of DNA origami and liposomes.
    When the concentration of aptamer is low (0.018, 0.069µM), DNA origamis hybridize with the surface of the liposomes relatively stablely. When the concentration is middle (1.8µM), more DNA origamis hybridizes with the surface and place stress on it. Then, liposomes become fragile and easy to be collapsed. When the concentration is high (6.9µM), some liposomes exist individually, and others form networks via aptamer and DNA origami complexes.


    According to this hypothesis, when the concentration of aptamer is 1.8µM, DNA origami collapses liposomes. Therefore, in the following experiment, we checked if DNA origami would collapse liposomes at this concentration of aptamer.


    2-3)Counting liposomes

    Purpose
    To see if DNA origami collapses liposomes, we counted the number of liposomes before and after adding DNA origami.

    Method
    For the sake of observation convenience, we mixed TR-DHPE (red fluorescent dye) with lipid (DOPC) and solvate (CHCl3), and made liposomes. The liposomal surfaces were dyed by TR-DHPE.
    Then we added aptamers at the final concentration of 1.8µM, and counted the number of liposomes with a fluorescent microscope.
    After counting, we put DNA origami and counted the number of liposomes again.
    Protocol




    ii)Flower micelle approach

    Experiment list

    The experiment necessary for realization of Flower micelle approach is following.
    1) Making liposome
    2) Confirming the hybridization of trigger and loop DNA
    3) Confirming the formation of loop structure by SPR
    4) Collapsing liposome


    1)Making liposome

    Purpose
    We make liposomes that are to be collapsed by flower micelle method.

    Principal
    We made normal liposomes made of DOPC and phase-separatied liposomes made of DOPC, DPPC and cholesterol.
    Phase-separated liposomes are liposomes made by several kinds of lipids. On the surface of phase-separated liposomes several kinds of lipids separate and the liposomes are formed by some layers.
    As the surface lipids of the phase-separated liposomes are not so changeable as the normal liposomes, It is considered that power produced by the hybridization of the loop and trigger strands reaches the liposome more effectively.
    So the phase separation liposome was used for experiments this time.

    Method
  • 1. Making DOPC, DPPC, and Cholesterol lipid Lipid
    1-1 Put 7.8 mg DOPC, 7.3mg DPPC , and 3.8mg Cholesterol into each microtube, and add 1ml CHCl3.
    1-2 Put it in a ultrasonic bath of 60 degrees Celsius for one hour.
    1-3 10mM DOPC, DPPC, Cholesterol lipid is made.

  • 2. Making phase-separated liposomes
    2-1 Mix DOPC,DPPC, and Cholesterol at the ratio of 1:1:1 to make phase-separated liposomes. In this experiment, mix 4µl DOPC (10mM), 4µl DPPC (10mM),4µl Cholesterol (10mM) and 88μl buffer well.
    2-2 Add 12µl Texas red (10μM)
    2-3 Dry the sample using Argon gas
    2-4 Hydrated the dried sample with by 100ml 1xTAE
    2-5 Put the sample in hot water for three hours. Then leave it at low temperature for one hour to let the surface lipid separate.

    Result
    As is shown in Fig.13, phase-separated liposomes were observed by a fluorescent microscope. They are basically multi-lamella liposomes.
    We confirmed the formation of phase-separated liposomes with a fluorescent microscope.

    Fig.13 Fluorescent microscope image of phase-separated liposomes

    Discussion
    Using the above-mentioned method, we successfully made phase-separated liposomes. However, they are multi-lamella ones and should be refined to be uni-lamella ones, by methods such as electroformation or droplet-transfer method.


    2) Confirming the hybridization of trigger and loop DNA

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

    Method
  • 1. Prepare three microtubes and put three kinds of trigger DNAs (10, 20, 40bases; 5µl, 100nM) into each tube.
  • 2. Add three kinds of loop DNAs (10, 20, 40bases; 5µl, 100nM) into corresponding tube (tube that contains trigger DNA of corresponding number of nucleotides) and leave them at normal temperature for approximately one hour.
  • 3. Add 6x loading buffer with the quantity of 20% of the samples.
  • 4. Make an acrylic amide gel.
  • 5. Load samples (including marker) into 10 lanes.
  • The electrophoresis was conducted with CV 100V for one hour.

    Result
    The result was shown in Fig.14.

    Fig.14 Stained gel image

    The lane of 20 base loop and trigger shows a strong band at different height from the band of only 20 base loop and trigger. As for the samples of 40 base, the result was the same.
    On the other hand, the lane of 10 base loop and trigger shows a band at the same height as the band of only 10 base loop. No band was seen in the lane of only 10 base trigger.

    Discussion
    The fact that the band of 20 base loop and trigger was at the different height from the band of only 20 base loop or trigger indicates that 20 base loop and trigger DNA hybridized and made a double strand. As the samples of 40 base showed the same result, we concluded that 20 and 40base loops and triggers hybridize at normal temperature.
    However, as for the samples of 10 bases, there was no difference between the two band height. Therefore, 10 base loop and trigger had not hybridized.
    It is estimated that no band was seen in the lane of only 10 base trigger because of some kind of mistakes. Therefore we do not take this into consideration.
    From the above, we find that the 20 and 40nt trigger hybridizes with a loop at normal temperature.


    3) Confirming the formation of loop structure by SPR

    Purpose
    To collapse liposomes by flower micelle method, we aim to attach many loop strands to the surface of liposomes.
    To achieve this, we adopt the same hybridization method via aptamers as used in i)Bending approach into liposomes: the aptamer has a complementary part to our loop strand and the loop strand is expected to hybridize with liposomes.
    We checked the hybridization of liposomes and aptamers, and that of aptamers and our loop strands.

    Principle
    As our loop strand is too small to observe with an AFM or a fluorescent microscope, we used an apparatus called SPR.
    SPR is a Surface Plasmon Resonance assay that estimates the weight of molecules attached to membrane surface, by the change of the reflection of the laser beam.
    If aptamer attaches to a liposome, and then loop strand attaches to it, SPR value increases after each step.
    We measured SPR value after each step of adding DOPC into liposomes, and loop DNAs into it.

    Method
  • 1. Inject 45µl DOPC (100mM) into SPR
  • 2. Inject 5µl NAOH to SPR in order to stabilize the point
  • 3. Inject 10µl aptamer (0.1µM) to SPR
  • 4. Inject 10µl loop DNA of 40 bp (0.1µM) to SPR
  • 5. Inject 10 µl trigger DNA of 40 bp (0.1µM) to SPR


  • Result
    The result was shown in Fig.15 below.

    Fig.15 The transition of SPR value

    As the first injection of aptamers caused no change of SPR value, we injected aptamers for two times.
    Fig 15 shows that SPR value increased after injecting aptamers and loop DNAs. Moreover, we should note that after injecting trigger DNA, some changes of SPR value were observed.

    Discussion
    Fig.15 shows the behavior of materials on the surface of liposomes. The increase of SPR value after injecting aptamers indicates that aptamers successfully combined with liposomes. Similarly, it is considered that loop DNAs combined with aptamers.
    Thus, we confirmed the formation of the loop structures on liposomes.


    4) Collapsing liposome

    Purpose
    It was tested if liposomes would be collapsed by adding trigger DNA.

    Principle
    Whether liposomes are collapsed or not can be decided by counting the number of liposomes before and after the trigger addition. As a control, we added the same amount of buffer instead of trigger. Liposomes are observed by a fluorescent microscope.

    Method
  • 1. Make liposomes with loop DNAs
    1-1 Mix 2µl liposome (0.2mM) with 2µl aptamer (10µM) at normal temperature
    1-2 Add 2µl loop DNA (20µM)

  • 2. Collapse the liposomes with the loop DNAs
    2-1 Add 2µl trigger DNA (20µM)


  • Result
    Fig.16 is the result of the sample added trigger DNAs; Fig.17, the sample of control experiment.
    Fig.16,17 Fluorescent microscope image of liposomes
    (Fig.16: sample added trigger DNAs, Fig.17: control)
    As it was difficult to count the number of liposomes in both cases, we did not count them.

    Discussion
    As we were not able to see a clear numerical change, we did not see whether liposomes were collapsed by this experiment.
    Two ideas why liposomes were not collapsed are come up:
    1. The lipid ratio for making liposomes was not appropriate. We should investigate the most appropriate and effective ratio for collapsing liposomes.
    2. Liposomes in this experiment were multi-lamella ones: Multi-lamella liposomes have some leaflets piled up. It is considered that more power is needed to collapse them. We would try other methods except the hydration method in future to make uni-lamella liposomes (which is relatively easy to collapse).
    Solving the above- mentioned problems, liposomes would be destroyed.

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