Contents

A tweezer B serves as a trigger to actuate Nanofingers to change its form between open and closed state. By optimizing the folding conditions, we successfully fabricate tweezer B initially with an open form. Folding of the tweezer B is carried out by applying a thermal ramp for about 1 hour. To characterize the folding outcome, TEM imaging is performed to observe the folding structure.

The switch of tweezer B from open conformation to closed conformation is achieved by introducing the fuel strand to the system. Based on a strand-displacement reaction, a fuel strand releases the antifuel that is partially complementary to the hairpin, resulting in a form switch to the closed conformation. The difference between these two conformations are indicated by the speed of migration in agarose gel electrophoresis, in which 3% agarose gels containing 0.1% ethidium bromide prepared at room temperature runs for 30 minutes at constant 100V and then is observed under UV.

The results are shown is Figure 2, which proves the oriented conformational change of tweezer B of which the size is about 300bp. The close of tweezer B gives it a more compacted structure which accounts for the faster migration speed in the electrical field. Therefore, we can conclude that the fuel strands successfully remove the antifuel strands with a strand-displacement reaction and change the form of tweezer B from open to closed. Different temperature (37℃ and 42℃) is adopted to find an optimizing condition for strand-displacement (lane 3 and lane 4) and the image shows that the reaction can proceed without obvious difference in efficiency.

The intact structure of Nanofingers is successfully folded after the origami experiment. Followed with a purification and concentration, we can finally see Nanofingers under AFM with a high yield. A magnified image of Nanofingers shown in figure 3c is corresponded to the model image in figure 3e, so that we can have a clearer understanding of its structure. In figure 3d, a height profile is given suggesting that Nanofingers measures about 15 nm in height, approaching our design 13nm, which confirms the successful folding. However, it is noticeable that the position of tweezer B (middle tweezer) differs from what we have designed in cadnano. It turns over outward the platform accounting for the low binding efficiency of the linker strand between the platform and the bottom of tweezer B. Yet it doesn’t affect the function of the whole device which can also be proved by the following experimental results.

We characterize the conformational change of Nanofingers using FRET between Cy3/Cy5 dye pairs.

By sequentially scanning the specimen with a fixed excitation wavelength (550nm, excitation wavelength for Cy3) and detecting the fluorescence in two channels, the emitted light of Cy3 and Cy5 can be clearly exhibited on images, showing us whether FRET occurs. When FRET occurs, Cy5 is excited showing a red color while sometimes there may exists a very weak green light probably caused by spectral bleed-through. In comparison, when FRET doesn’t occur, the emission light of Cy3 and Cy5 can both be detected at the same point and we can see yellow overlap spots in the merged image. We number yellow spots in FRET image and non-FRET image and give an evaluation on the difference between the two different status.

As shown in Figure 4b, in the three images (top) of the first sample, red light takes advantage over green light and only one yellow spot is collected in the field of view, demonstrating high efficient FRET of Nanofingers. In contrast, when the second sample is observed, red light declines while green light is enhanced, resulting in the significant increase in the yellow spots (10 yellow spots). As shown in Figure 4c, we achieve the highly significance difference (marked on “**”) between these two samples by analysis of data with histogram. Therefore, we can draw a conclusion that an efficient conformational change takes place in Nanofingers by adding fuel strands.

We also demonstrate the FRET effect by measuring the fluorescent intensity changes of Cy3 and Cy5. Initially, tweezer B on the assembled Nanofingers is open and the two tweezers A are closed, which exhibits a lower Cy3 signal and a higher Cy5 signal due to FRET effect. When fuel strand is added, antifuel strand is removed from the hairpin inside tweezer B, making it change to its closed conformation. Through linking strands, tweezers A are dragged open motivated by tweezer B. In this state, the distance between Cy3 and Cy5 is too large for efficient FRET, resulting in a decrease in fluorescent intensity for Cy5 and an increase for Cy3.

Depicted in Figure 5, by adding the fuel, FRET-state Nanofingers switches to non-FRET state, causing an increase in Cy3 signal and slight decrease in Cy5 signal. Removing the fuel strands by addition of the antifuel strands drives Nanofingers back to the initial state, resulting in the decrease in Cy3 signal and slight increase in Cy5 signal. This cycle is repeated twice. During the whole process, the fluorescent intensity decays gradually due to photo bleaching. When measuring Cy5 signal, the high concentration of fuel and antifuel strands significantly affects the detection, resulting in a loud noise in the system and slight change in Cy5 signal.