Biomod/2012/UTokyo/UT-Hongo/Intro

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The University of Tokyo

Contents

Focus

Fig.1 Schematic of our goal in the long run
Fig.1 Schematic of our goal in the long run

Our focus was to use DNAs to make a device that would complement the defects of enzymes, and therefore allowing expansion of the areas of enzyme application.

Enzymes are widely used for their specific binding and catalytic abilities. However, it is difficult to fully exploit the advantages for two reasons. The first reason is that they can easily be deactivated by external factors such as the presence of proteases. Also, even though it is getting easier with the growth in bioengineering, it is still difficult to modify the structure of the enzyme for purposes such as addition of a new functionality. This is because the structure of enzyme is delicately controlled, and any artificial modification could ruin the structure and thereby the original function. These are some of the difficulties in using enzymes.

We focused on the DNA molecules for they are the optimum materials to complement such difficulties. DNAs are stable in various environmental conditions, and by making a rigid structure, it can also be stable against DNAases. The structure of DNA can also be easily adjusted and modified to add new functionalities. By combining enzymes and DNAs, we sought to make a nanodevice that can be used for a wider field of application.


Idea of DNA Shell

Fig.2 Schematic of the "Shell Mechanism" in comparison with the natural shellfish
Fig.2 Schematic of the "Shell Mechanism" in comparison with the natural shellfish

To combine DNA and enzyme, we need a mechanism to connect the two components. The first idea that came to us was the "Shell mechanism". Just like the ordinary shellfish that we see in nature, our DNA shell captures the target by embracing it with the two origami surfaces.

In order to realize this idea, we chose to capture streptavidin by functionalizing the DNA shell with biotin as a model case. Streptavidin is an enzyme known to bind very strongly with biotin. Since streptavidin are known to be in the form of tetramers, we can use the biotin modified DNAs to capture the streptavidin from the two sides using the "Shell mechanism".


Functionalities Exhibited

Through the shell structure and some further modification, we proposed several functionalities that could not be possible with DNA or enzyme individually. They are...

  1. Molecular Sensing
  2. Substrate Protection
  3. Attachment to solid surface

The key to these functionalities was the characteristics in the shell structure, the feasibility of DNA modification of the shell, and the stability of DNA molecules. Possible fields of applications for these functionalities are written on Progress & Beyond page.


Highly Sensitive Detection

Fig.3 Scheme of the detection mechanism. The difference in the flourescence is much larger for DNA shell compared to the conventional fluorescent DNA devices
Fig.3 Scheme of the detection mechanism. The difference in the flourescence is much larger for DNA shell compared to the conventional fluorescent DNA devices

The molecular detection with higher sensitivity could be achieved by the DNA Shell. This is because of the capturing mechanism. Conventional DNA devices only used one fluorescent molecule and quencher molecule. This way, the difference in the fluorescence is very small. However, since the DNA shell captures the molecules using the DNA surface as a whole, we can add more fluorescent and quenching molecules. With more of each molecules, we can expect a larger change in the fluorescence when the device captures the enzyme. The difference of the DNA shell and the conventional DNA device could be easily understood by comparing it with the ease of detection of a lighthouse turning its power off and a little light bulb turning off.


Enzyme Protection

Fig.4 The use of DNA shell for protection of enzymes. The size-selective feature of proteases is the key to our methods
Fig.4 The use of DNA shell for protection of enzymes. The size-selective feature of proteases is the key to our methods

Just like shellfish uses their shells for protection in the nature, we can use the DNA shell to protect the enzyme inside. When an enzymes gets decomposed, it is usually done by proteases, another group of enzymes that break down enzymes. Proteases decompose the enzymes by capturing it inside its structure and using some strong chemical reagents to destroy it.

By using the DNA shell such decomposition could be prevented. The key in preventing the decomposition is not to let proteases take in the enzyme. When the enzyme is captured inside the DNA shell, the combined structure would be too big for the protease to capture, and thereby the decomposition will not occur.


Attachment to Solid Surfaces

Fig 5. The difference of when DNA shell is attached to a surface and when it is not. Attachment to a solid surface will definitely give a boon when thinking of practical application.
Fig 5. The difference of when DNA shell is attached to a surface and when it is not. Attachment to a solid surface will definitely give a boon when thinking of practical application.

Attachment to a solid surface is very important when we think of practical application of the DNA shell. Imagine when enzymes are used inside a reactor. When the enzymes are floating in the solution, they will also be washed away with the flow. However, by attaching it to a solid surface, we can keep the enzyme from being washed away. This way, not only can we keep the reactor working longer, but can obtain the product without the DNA shells as the impurities. In another words, we could use DNA Shell as a heterogeneous catalysis by attaching it to a solid surface.

Usually, it is sometimes difficult to attach enzymes onto a solid surface. The reason is because the structure of the enzymes could change easily damaged by attachment to solid, and thus leads to the deactivation. However, by using DNA shell, we can neglect such problems because DNA strands are strong against environmental changes.

We experimented this feature using the microfluidics device, a device which has a high surface to volume ratio, and therefore is a practical candidate when making reactors that uses enzymes as catalysis.

Goal for BIOMOD

Through the dicussion above, as a goal for BIOMOD, The University of Tokyo, Team Hongo chose to

  1. Realize the DNA shell structure
  2. Confirm the three possible functionalities through experiments

Through these goals, we tried to make a new DNA device which can complement the problems of enzymes.

Conceptual Blueprint of the Structure

Fig.6 Schematic of DNA Shell in detail
Fig.6 Schematic of DNA Shell in detail

We designed our DNA shell so that all the functionalities described above could be realized. The structure consists of three DNA origami domains; the fluorescent domain, the quencher domain, and the attaching domain. The capture takes place by using the first two domains. In the center of the fluorescent and quencher domain, there is the area for attaching DNA staples that has the bonding ability. Fluorescent and quencher molecules are attached in a circle around the area for bonding DNAs. The links between the domains are made loosely, with two one double-helix. This is because, if otherwise, the structure may be too rigid for the structure to fold in two for the capture.


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