Biomod/2014/Kashiwa/Trials

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EARLY TRIALS  

 Design

In order to achieve the project goal, we designed two constructs using DNA origami; the Receptor for the sensing system and the Motor-Monomer for the moving system.


 The Receptor

The Receptor was developed to recognize an external signal on the liposome surface upon which it releases the Polymerization Initiator inside. For this, we designed a pair of hetero-units for the Receptor, the Receptor E and the Receptor A. They dimerize when an external signal is recognized and cause the release of the Polymerization Initiator (In this case, a single-stranded DNA).

Requirements

To develop the Receptor, the following three mechanisms have to be considered:

1. Embedding of Receptor into the liposome
2. Recognization of the external signal
3. Release of the Polymerization Initiator internally

Structure

We used OCK structure for trial experiments (http://openwetware.org/wiki/Biomod/2013/Todai). We attached aptamer sequence(red) and Initiator(orange) to OCK structure; composed of floater domain(blue) and membrane integrating domain(yellow). Green staple has connecting role between Receptor and Initiator.

Strategy

The details of our strategy to fulfill the requirements are described below.


1. Embedding of Receptor into the liposome

The natural Receptor often just sticks on the outside of the liposome and rarely penetrates it. Therefore, we modified the Receptor with cholesterol so that it penetrates the liposome and can access the interior.


2. Recognization of the external signal

We used Thrombin as an external signal in this experiment. Thrombin, a serine protease, has specific sites where different DNA aptamers bind. We inserted thrombin-binding DNA aptamer sequences into the Receptor hetero-units. Furthermore, we bound a staple strand to each hetero-unit, two strands to be complementary to each other.

Therefore, when the aptamers in the Receptor hetero-units bind to the binding sites of thrombin, the hetero-units join together to form a dimer. We should keep in mind that we have to optimize the thrombin concentration; an excess of thrombin causes errors because of the Receptor E and the Receptor A binding to the separate thrombin.


3. Release of the Polymerization Initiator

We designed the Polymerization Initiator as a single-stranded DNA which is partially complementary to a specific domain of the Receptor E. When the Receptor hetero-units form a dimer, the strand displacement occurs to form a more stable DNA duplex and the initially-bound ssDNA is released, which works as the Polymerization Initiator. 



 The Motor-Monomer

The Motor-Monomer was developed to begin the polymerization when it caught the Polymerization Initiator. For this, we designed two types of the Motor-Monomers, Motor-Monomer X and Motor-Monomer Y, which have closed rings; when the Polymerization Initiator is caught, the Motor-Monomers join to form the Motor-Polymer by opening their ring structures one after another.

Requirements

To develop the Motor-Monomer, the following two requirements have to be considered:

1. The Motor-Monomers should not polymerize without the Initiator.
2. The Motor-Polymer should be stiff enough to deform the liposome.

Structure

Provisory structure of Motor-Monomer for experiments. We attached six connecting staples(orange) to Flip-tile(blue). The length of connecting staple was determined for making ring formation.¹

Strategy

The details of our strategy to fulfill the requirements are described below.


1.Controllability

In order to simplify the condition of ring structures, we designed the Motor-Monomer with a ring structure at first. Strand a, a’, b and b’are ssDNAs embedded in the ends of the Motor-Monomer X and Y. Strand a-b pair and a'-b' pair are partially complementary. In contrast, strand a-a' pair and b-b' pair are completely complementary. By using these staples, Motor-Monomers becomes hetero-polymer. It helps us prevent the Motor-Monomers from polymerizing before building ring structures, and increase the stiffness of the Motor-Polymer because it enables the Motor-Monomers to get close to each other.

However, the persistence length of the ssDNA is only a few nanometers while the length of Flip-Tile is 34 nm. Therefore, it is difficult to make ring structures after we embed ring staples into the ends of the Motor-Monomers. To resolve this problem, we introduced short ssDNAs (named cover staples), which were partly complementary to the ring staples to form double strand DNA, which are much more rigid structure (the persistence length of dsDNA is about 50 nm). Take everything into consideration, we mixed the ring staples and the cover staples at first. Then, we mixed this complex with the Flip-tile structure, and could embed the ring structure into the Flip-Tile structure. Subsequently, we dissociated the cover staples from ring structure by raising the temperature to the melting temperature of the cover staples. In this way, we could get the Motor-Monomer which had a closed staple ring structure.

Next, when the Initiator strands are released, the Monomer opens its ring via strand displacement. Then, the free end of the Monomer forms a DNA duplex by a displacement with one end of another Monomer and the other end becomes free. In this way, the Monomers polymerize one by one.



2.Bending stiffness

Our initial approach was to design three closed rings in the Motor-Monomer to develop enough stiffness by limiting flexibility. Subsequently, we found that three-rings-Monomers are likely to form the branched Polymer, based on probability theory. Therefore, we try a new approach according to the reported reference (Iinuma et al)².

  • Firstly, we planed to introduce concavo-convex surface to ensure the geometry specificity of the adherend. Secondly, additional short sticky strands (a few bases) were introduce to strengthen the binding stability.



 

 Approaches

EXPERIMENTS

The Receptor

 Direction

We planned to do experiments in several steps. First, we design the Receptor. Then, the following experiments are conducted in parallel.

• Form the Receptor dimer by using thrombin.
• Emit the Polymerization Initiator by demerization.
• Embedding of Receptor on the liposome.



Finally, after success in the experiments, we combine them to attain the dimerization and the Initiator emission on the liposome.



 Experiments

 Folding the Receptor


Folding of the Receptor was confirmed by TEM and 1% agarose gel electrophoresis analysis.
The folded structure of the Receptor was clearly appeared in the TEM image.

 


 Forming the Receptor dimer

In this experiment, the dissociation constant (Kd) of thrombin and thrombin-binding aptamers was estimated. The purpose is to confirm the binding ability of each Aptamer (Aptamer A and B) with a thrombin before checking dimerization of the Receptor hetero-units. Binding of the aptamers with thrombin was analyzed with Native-PAGE.



We could confirm that aptamers bind to thrombin based on these two results. First, there are bands on the upper of that of aptamer's in the lanes (added aptamers 20 or 10 times as much as thrombin) of the left electrophoresis figure. Second, in fig.2, the bands of aptamers are weaker in the lanes (added thrombin 50 or 25 times as much as aptamers) than the other lanes, and we attributed these results to the formation of the complexes of thrombin and aptamers. In addition, the Kd of aptamers and thrombin is estimated about 50-300nM by measuring the intensity of band with the software (Image J).

 


 Emitting the Initiator

We confirmed whether the strand displacement mechanism was worked, and confirm the release of Polymerization Initiator. Also, we optimized the sequence by examine the effect of mismatch ratio of the strands.

We used the following abbreviation;
J: Non-modified single strands
J’: Biotin modified single strands
(J is completely complimentary to J’)
Ini: Initiator (The percentage of mismatch to J is 5, 20 and 40%)
Cy5-modified Streptavidin (SA)




Figure native-PAGE stained by EtBr.
J: Non-modified single strands
J’: Biotin modified single strands. Completely complement to J.
Ini XX%: Initiator. Partially complement to J with XX% mismatch introduced.
SA: Cy5-modified streptavidin.




Figure The ratio of released Initiator to the whole Initiator, with 5 % and 20 % mismatch introduced sequence. The result showed that little Initiator was released with this condition.

Based on this experiment, the displacement of hybridized strand, and the release of Initiator was confirmed. Initiators were hardly released when the percentage of mismatch is 5 %, while with 20 % mismatch Initiators were released. For 40 % mismatch sequence, the band of released Initiator and that of original Initiator - J hybridized duplex were hardly distinguishable. To overcome this problem, it might be better to use fluorescently labeled strands.

 



 Embedding on the liposome


In order to certify the penetration of the Receptors to liposomes, a flotation assay was conducted.

In preparation for a floation assay, cholesterol oligomers were hybridized to the Receptors for penetration. The hybridization was confirmed by 1% agarose gel electrophoresis analysis.





In flotation assay, we divided mixture with the Receptors and the liposomes to a few fractions, each of them was analyzed by fluorescence spectrophotometer and 1% agarose gel electrophoresis. This assay showed the distribution of liposomes and the Receptors, therefore we can distinguish the Receptor embedded into the liposomes from the free Receptors floating in solution.





The fluorescence intensity of NileRed in each fraction (NileRed dyes the membrane of liposomes)

The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3.








The ratios of the Receptor in each fraction were analyzed by the density of band.

In sample1 (cholesterol + / liposome +), the ratio of the Receptor in fraction3 was a bit larger than that in fraction2. In contrast, in sample 2, 3 and 4, the ratio of the Receptor in fraction3 was smaller than that in fraction2. As liposomes were mostly seen in fraction3 in both sample 1 and 3, and the ratio of the Receptor in each fraction was different only in sample1, we concluded that this difference was caused by penetration of the Receptor to liposomes.





 Next Steps

In these experiments, we achieved to construct the Receptors and insert them into the liposomes. The very basics of others, formation of the Receptor dimers and emission of the Initiator, were also confirmed. Therefore, we are planning to approach the ones following for next steps.

• Confirm the more advanced mechanism of forming the Receptor dimers and emitting the Initiator.
• Examine conditions for dimerization of the Receptor heterounits on the liposomes, may need changes in design of the Receptor.

 



The Motor Monomer

 Direction


We planned to do experiments in several steps. First, we design the Motor-Monomer and assay the formation of a simple Motor-Polymer. Then, the following experiments are conducted in parallel.

• Control the initiation of the polymerization (forming ring structures and ring-opening polymerization).
• Put the Motor-Monomers into the liposome.


Finally, after success in the experiments, we combine them to attain the polymerization in the liposome.





 Experiments

 Folding the Motor-Monomer

The assembly condition of the Motor-Monomer was optimized with concentration of MgCl2, annealing temperature and the ratio of staples to scaffold. The resulting structure was analyzed by agarose gel electrophoresis.





Multimers (e.g. dimers) appeared at lower temperature, so optimum temperature of annealing was 55.0℃.







(Annealing temperature : 55.0℃, M13 : staples = 1 : 2)
The band of monomer at 16mM is bending and its migration distance is different from the one of MoN. It shows that the Monomers were not annealed correctly at 16 mM. There seemed to be little difference at other concentration, so optimum concentration of MgCl2 was the range 10 to 14mM.




(Annealing temperature : 55.0℃, MgCl2 : 10mM)
At the ratio of 3 and 4 a band of a monomer is weak and migrating distance was shorter than the one of monomer. As the graph showed, the ratio of dimer to monomer was low in lane 1 and 2, so optimum ratio of staples to scaffold was 2.

Folding of the Motor-Monomer was corroborated by agarose gel electrophoresis analysis. The folding, however, was not verified by TEM. We considered it was because of hollow structures of the Motor-Monomers.





 Forming the Motor-Polymer

MoN: The Motor-Monomers with no arm    negative control: mixture of both inactivated MoX and MoY


In this experiment, the effect of the number of arm on polymerization was examined. Two samples, one has three arms extruded from the body and the other has one, was compared. The Monomers comprise two similar structures, the Motor-Monomer X and the Motor-Monomer Y. The polymerization was confirmed by mixing the Monomer X and the Monomer Y.

The ratio of dimers of three arms monomers roughly increased with time, while the ratio did not change significantly for one arm monomers. It confirmed the polymerization of the Monomers with three arms.




The Monomer concentration was optimized.

The ratio of dimers was high at high monomer concentration of three arms monomers, while for one arm monomers, the ratio was not different from that of monomer only condition. It meant Polymerization of three arms monomers proceeds efficiently at high concentration of monomers in solution.

Reference

1.M. Douglas et al, “A logic-gated nanorobot for targeted transport of molecular payloads”, Science, 2012 Feb 17; 335(6070): 831-4.
2.Science. 2014;344(6179):65-9.Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT.Iinuma R, Ke Y, Jungmann R, Schlichthaerle T, Woehrstein JB, Yin P. Nature. 2006 16;440(7082):297-302. Folding DNA to create nanoscale shapes and patterns. Rothemund PW.
Nat Methods. 20118(3):221-9. A primer to scaffolded DNA origami.Castro CE1, et al.
Nat Nanotechnol. 2008 3(7):418-22. Self-assembled DNA nanostructures for distance-dependent multivalent ligand-protein binding. Rinker S, Ke Y, Liu Y, Chhabra R, Yan H.
J Mol Biol. 1997 272(5):688-98. Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. Tasset DM1 Science. 2012 335(6070):831-4. A logic-gated nanorobot for targeted transport of molecular payloads. Douglas SM1 Science. 2012 16;338(6109):932-6. Synthetic lipid membrane channels formed by designed DNA nanostructures. Langecker M1, Kilchherr F, Kim DN, Shiao EL, Wauer T, Wortmann P, Bathe M, Dietz H.

© 2014 UTokyo Chem & Bio

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