Design

Enzymatic device

In this page, we explain the device using DNA and enzymes. First, we overview the outline of mechanism.

The device includes three processes, which are Reading process, Releasing process and Updating process. In Reading process, Input DNA gives instructions of the releasing order to the device. With the first instruction, a corresponding Signal is activated and transmitted to next processes. In Releasing process, Output is released by the Signal from the previous process. After releasing, Updating process starts. In the Updating process, Input is renewed by deleting the first instruction on it. By cycling these three processes, single-stranded DNA output are generated in order, whose sequence is coded in Input DNA.

Reading process

In the following, we explain the details of the three processes. For the Reading process, we need Templates, Transducer, and Gates as shown below.

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Step1. Templates hybridize with Input, which results in Input/Template complex.
Step2. Polymerase elongates Input at its 3' end. We introduce unpaired bases at 5' end of the Template to prevent undesired elongation.
Step3. Nickase cleaves elongated Input.
Step4. Polymerase elongates remaining Input from 3' end again with its strand displacement activity. As a result, Signal is released.

Repeating Step3 and Step4, single stranded DNA (Signal) is amplified. The Signal will be transmitted to the next processes: Releasing and Updating processes.

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Releasing process

Following chemical reactions represent the Releasing process. It is conposed of Signal, Transducer, and polymerase.


Step1. Signal hybridizes with a single stranded part of Transducer.
Step2. Polymerase elongates Signal from 3' end.
Step3. Output DNA is released by strand displacement activity of polymerase.

This process takes place just after reading process because the toehold region of Transducer is slightly longer than that of Gate (for detail, see Materials&Methods).

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Updating process

Updating process is composed of Input/Template complex, Signal, and Gate.

Step1. Signal hybridizes with a single stranded part of Gate.
Step2. Polymerase elongates DNA from 3' end.
Step3 .Single stranded DNA (Updater) is released by strand displacement activity of polymerase.
Step 4.Updater hybridizes with Input/Template complex to form a recognition site of restriction enzyme.
Step5. Template-A is released from Input by strand displacement.
Step6. Restriction enzyme cleaves duplex of Updater and Input/Template complex. Consequently, Input is updated for the next cycle.

This cycle is repeated again and again until all the instructions written in Input are executed. Eventually, the Outputs are released in programmed order.

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Enzyme-free device

In this page, we explain the mechanisms of Enzyme-free device (using only DNA).Enzyme-free device is designed based on seesaw gates [1] which is capable of cascading strand displacement reactions. Components of the device are Input, Trigger, Gate, Transducer, and Fuel.


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Like Enzymatic device, the order of releasing output is coded in Input, while other DNA components are utilized for cascading the reactions. Cascading reactions are implemented by the following steps.


Step1. Trigger hybridizes with Input.
Step2. Signal is released by strand displacement reaction.
Step3. Signal hybridizes with Transducer. This step takes place just after Step2 because the toehold region of Transducer is slightly longer than that of Gates.
Step4. The first Output is released by strand displacement.
Step5. Remaining Signal starts to hybridize with Gate.
Step6. By consuming Fuel, a new Trigger is released for the next reaction. Since this is catalytic reaction, sufficient amount of trigger is released from small amount of Signal. (details of catalytic reaction by consuming Fuel)
Step7. The new trigger hybridizes with a single stranded part of Input.
Step8. A new Signal is released by strand displacement reaction.

The device repeats this cycle until all Outputs are released. As well as Enzymatic device, programmability is an important feature of the device.


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Reference

[1] L. Qian, E, Winfree, Science 332 (6034) (2011) 1196-1201