We designed “Linker-output complex”(Fig. 1) to make it release a DNA which connects gold nanoparticle and microbead caused by “DNA signal”. “Linker-output complex” has a single stranded sticking out end which is composed of five base pairs complementary to a part of “DNA signal” strand(red portion in the Scheme. 1 and Fig. 1 below). This end is called “toehold” and triggers strand displacement reaction.This reaction proceeds as Scheme. 1 . First, a site of “DNA signal” strand which is complementary to the toehold binds to the toe hold(Scheme. 1. (b)). Based on this binding, “DNA signal” starts detaching “Linker strand”.Finally, “DNA signal” forms a duplex and release “Linker strand” completely (Scheme. 1. (d)).This process from (a) to (c) is an equilibrium.However, once “Linker strand” is released(Scheme. 1. (b)), state (d) doesn’t go back to state (c) because “Linker strand” doesn’t have a toehold to bind again(blue portion in the Scheme. 1 and Fig. 1 below).
Fig. 1. Sequence of Linker-output complex
To make DNA signal release only when the amount of UV exposure exceeds a certain level, we utilized Threshold complex. The sequences of Threshold complex and Linker-output complex are very similar; both complexes reacts with DNA signal. However, as you can see in Fig. 2 , the two complexes differ in their toehold lengths. Namely, the toehold of Threshold complex consists of nine bases, and that of Linker-output complex consists of five base pairs. This difference causes the difference in their affinity for DNA signal and makes Threshold complex react earlier than Linker-output complex.
Assuming that Threshold complex reacts completely ahead of Linker-output complex, DNA signal starts to react with Linker-output complex and make it release Linker strand only when the signal’s amount exceeds the amount of Threshold complex. This means that the concentration of Threshold is just the threshold value of the whole system, so all you need to change the threshold is to change the concentration of Threshold complex.
Fig. 2. Sequence of Thresholding DNA(above) and its mechanism(below)
 Lulu Qian, Erik Winfree, ;J. R. Soc Interface, 91125 (2011)
To achieve Threshold processor, we conducted the following two preliminary experiments.
1. Reaction between DNA signal and Linker-output complex
To confirm the strand displacement reaction between DNA signal and Linker-output complex, we utilized Fluorescence quenching mechanism. As described in Fig. 3 , our Linker-output complex has a FAM (a kind of fluorescent molecule) and a BHQ1 (a kind of quencher molecule) at the end of both strands. Placing FAM and BHQ close this way causes an energy transfer called FRET (Fluorescence Resonance Energy Transfer), in which the original wavelength of FAM is no longer observed. This quenching ceases when strand displacement happens and the two molecules separate. Thus, we can confirm the strand displacement as the shift of fluorescence intensity.
Fig. 3. Reaction scheme of DNA signal and Linker-output complex
Firstly, we prepared Linker-output complex and then added DNA signal to cause strand displacement reaction. The concentration of Linker-output complex is 0.2μM, and the concentrations of DNA signal are respectively 0μM(0%), 0.05μM(25%), 0.10μM(50%), 0.15μM(75%)、0.20μM(100%). Then we measured fluorescence intensities of the samples in each concentration at the wavelength of 520nm, converted them into strand displacement rates and described in the graph below. This graph shows that Linker-output complex steadily reacts with DNA signal as the concentration of DNA signal becomes higher.
Fig. 4 . Transition of the reaction rate of Linker-output complex
2. Reaction between DNA signal and Threshold complex
We used the same method as the experiment1 to confirm the strand displacement between DNA signal and Threshold complex. The fluorescent molecule here is Cy5 and quencher molecule here is BHQ3 (Fig. 5).
Fig. 5. Reaction scheme of DNA signal and Threshold complex
The graph below shows the result of the experiment. The concentration of Threshold complex is 0.2μM, and the concentrations of DNA signal are respectively 0μM(0%), 0.1μM(50%), 0.15μM(75%), 0.2μM(100%)、 0.25μM(125%) and 0.3μM(150%). This graph also shows the steady reaction between Linker-output complex and DNA signal.
We attached different pairs of fluorescence molecule and quenching molecules to Threshold complex (Cy5 and BHQ3) and Linker-output complex (FAM and BHQ1), and observed those strand displacement reactions with DNA signal at each concentration by fluorescence measurement (Fig. 6).
In this experiment, we verified that the difference of strand displacement reaction rate brings about a certain threshold. The final concentrations of Linker-output complex and Threshold complex are 0.2μM and 0.4μM respectively, and the final concentration of DNA signal is every 0.1μM from 0μM to 1.0μM.
We calculated the reaction rate of each strand displacement by converting and standardizing fluorescence intensity: in each reaction, we regarded the lowest fluorescence intensity observed as reaction rate 0%, and the highest fluorescence intensity observed as reaction rate 100%.
From this result, we confirmed that DNA signal primarily reacts with Threshold complex, and then reacts with Linker-output complex.
In this experiment, DNA signal reacted with Linker-output complex even during its reaction with Threshold complex. Ideally, DNA signal reacts with all threshold complexes first, and then starts to react with Linker-output complex, so this result cannot be regarded as a good one.
We estimate that the result could be improved by designing a better sequence of Linker-output complex.
Fig. 6. Threshold processing of the amount of DNA signal