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To complete our goal, it is necessary to develop a system that releases output liposome in order. The liposome encapsulates taste substances and is immobilized on substrate. The order of output is coded on input DNA. Following two different approaches are proposed.
1st Approach: Enzyme system
2nd Approach; Enzyme-free System

1st Approach: Enzyme system

This system uses DNA as an input and following reaction are driven by enzymes such as polymerase, nickase, and restriction enzyme. These enzymes can be regarded as hardware while DNA serves as software. In this system, there are three processes shown in Fig.1. Before the addition of input DNA, some sets of DNA molecule (Templates, Gates) and enzymes are mixed in advance.

1. Amplifying process: DNA polymerase amplifies key-DNA for next 2nd and 3rd process.
2. Releasing process: Key-DNA releases the liposome that encapsulates taste substances.
3. Updating process: Restriction enzyme updates the input-DNA to repeat next circle.

Following section explains the enzyme system when the input DNA encodes “ABC”.

Fig.1 Schematic explanation of Enzyme system

1. Amplifying process

In this process, DNA polymerase creates key-DNA (in this case A1) for next processes. Then, key-DNA A1 is amplified by polymerase, nickase, and templates when input is added.(Fig.2) The sequence of template-A is 5’-A1 A0-3’. The strand A0 is complementary to A0. Furthermore, Input DNA that encodes “ABC” has a sequence of 5’-C0 x B0 x A0-3’.

The details of this process is shown as follows.

When input DNA is added, the strand A0, B0, and C0 in the templates hybridizes to strand A0, B0, and C0 in the inputs respectively (we name the hybridized structure “Complex”)(①). Then, polymerase extends the input from 3’ end synthesizing new sequence A1 that is complementary to the template DNA(②). After that, the nickase cleaves strand between A0 and A1 by recognizing the sequence in A0(③). Then, polymerase works at the gap created by the nickase thereby DNA displaces the domain A(④). Repeating ③,④, the strand A1 is amplified.

This amplified strand will become an input of following process 2nd and 3rd.

Fig.2 Schematic explanation of the amplifying process

2. Releasing process

This process releases the liposome-A when it accepts the strand A1 from amplifying process.(Fig.3) The liposome is immobilized on substrate in advance. To immobilize that, cholesterol-modified DNA (output-A) and thiol-modified DNA can be used. We can fix thiol-modified DNA onto gold plate. When output-A attached on liposome hybridizes to thiol-modified DNA, liposome is immobilized on substrate.

The details of this process is shown as follows.

After the amplifying process, strand A1 hybridizes to thiol-modified DNA on substrate. Polymerase synthesizes new DNA from the 3’-end of A1(⑤), separating existing hybridization(⑥). Eventually, liposome-A is released with output-A from the strand A1.

Fig.3 Schematic explanation of the releasing process

3. Updating process

In this process, the code “A” on the complex is updated to next code (in this case “B”) by gate-A, polymerase, and restriction enzyme.(Fig.4) By key-DNA A1 from amplifying process, gate-A creates signal-A whose sequence is 5’-A0 x α-3’. The domain “x” is recognition site for restriction enzyme.
Signal-A forms the recognition site for restriction enzyme when it hybridizes to the complex. After that, restriction enzyme cleaves the complex. This cleaving means that the code on input updated.

The details of this process is shown as follows.

After A1 hybridize to Gate-A, polymerase extends A1(⑦) and displace a signal DNA(⑧). When the signal DNA hybridizes to the complex, a recognition site for the restriction enzyme is formed(⑨). Due to the recognition site, restriction enzyme cleaves the complex, forming a next complex(⑩).

The code of the complex is updated to B,C for next repetition.Regardless of the numbers and kinds of domains, the orders of outputs only depend on encoded information in the input.

Fig.4 Schematic explanation of the updating process

2nd Approach; Enzyme-free System

The purpose of Enzyme-free System is to release outputs by using Seesaw Gate in order which is written on an input. The system has three processes shown in Fig.5. When the input DNA encodes “ABC”, reactions in this system are mainly divided into three processes as follows.

1. Key-DNA releasing process: key-DNA is released by hybridizing input and trigger.
2. Output releasing process: key-DNA releases the liposome that encapsulates taste substances.
3. Amplifying process: Fuel amplifies the trigger which is attached to the input to repeat the next cycle.

Fig.5 Schematic explanation of Enzyme-free system

The DNA prepared beforehand is shown in Fig.6. Trigger (T1), Gate (Ga, Gb, Gc), fuel (Fa, Fb, Fc), and liposome (La, Lb, Lc) are mixed in solution in advance. The sequence "t", "a", "b", and "c" are five bases. In addition, toehold of liposome is two bases longer than those of gates.

Fig.6 Preparation of Enzyme-free system

1. Key-DNA releasing process

The purpose of this process is to produce key-DNA for the next 2nd and 3rd process (Fig.7). In this process, trigger and input make key-DNA. To release output A, B, and C, input has all key-DNA that correspond to output respectively. First, a sequence “t” in trigger hybridizes with the input which has complementary sequence “t”. Next, trigger is replaced with key-DNA at “x”. Final, key-DNA is released because the sequence “a” is short. Key DNA causes the next reaction.

Fig.7 Schematic explanation of the key-DNA releasing system

2. Output releasing process

After key DNA is released in process 1, it reacts with La (Fig.8). With this reaction, output DNA attached to Liposome is released. This reaction occurs faster than that of key DNA and Ga because toehold in La is two-base longer than that in Ga.

Fig.8 Schematic explanation of the output releasing system

3. Amplifying process

After process 2 has finished, the remaining key DNA starts to react with Ga (Fig.9). This reaction generates T2 and a by-product DNA. Fa is added to produce key DNA again, by reacting with by-product. Using Fa, T2 is repeatedly produced unless all Fa is consumed. Next, amplified T2 reacts with I2. This reaction begins the next cycle of producing second liposome and T3. In the end, reaction of T3 and I3 release third Liposome in the same way.

Fig.9 Schematic explanation of the amplifying system
(C)Copyright Biomod 2014 Team Sendai

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