A Quick-responsive DNA Nanotechnology Device for Bio-molecular Homeostasis Regulation

Physiological processes such as metabolism, cell apoptosis and immune responses, must be strictly regulated to maintain their homeostasis and achieve their normal physiological functions. The speed with which bio-molecular homeostatic regulation occurs directly determines the ability of an organism to adapt to conditional changes. To produce a quick-responsive regulatory system that can be easily utilized for various types of homeostasis, a device called nano-fingers that facilitates the regulation of physiological processes was constructed using DNA origami nanotechnology. This nano-fingers device functioned in linked open and closed phases using two types of DNA tweezers, which were covalently coupled with aptamers that captured specific molecules when the tweezer arms were sufficiently close. Via this specific interaction mechanism, certain physiological processes could be simultaneously regulated from two directions by capturing one biofactor and releasing the other to enhance the regulatory capacity of the device. To validate the universal application of this device, regulation of the homeostasis of the blood coagulant thrombin was attempted using the nano-fingers device. It was successfully demonstrated that this nano-fingers device achieved coagulation buffering upon the input of fuel DNA. This nano-device could also be utilized to regulate the homeostasis of other types of bio-molecules.


Tweezers B
Blue: The hairpin of B8 Red: The complementary sequence of the hairpin

Verification of scaffold DNA
3.2.1 the concentration of ssDNA achieved was measured by nucleic acid analyzer.

3.2.2
To verify whether the ssDNA extracted from M13 phage is right the material needed, the ssDNA was transformed into E. coli again and sequenced. The sequencing result and blue spot both confirmed the DNA material.

DNA Origami
Before starting to fold nano-fingers, Correct structure of tweezers B is firstly to be constructed to verify the feasibility of structure design.
3.3.1 Folding of tweezers B 50uL reaction system PCR procedure 3.3.2 Folding of nano-fingers 50 μL reaction system PCR procedure

The purification and concentration of tweezers B and nano-fingers
To ensure successful folding, the staple strands were added in a 10-fold higher concentration than the scaffold.
For purification of tweezers B or nano-fingers from the excess staple strands, a filtration step was carried out after folding. Amicon Ultra-0.5 Centrifugal Filters with a pore size of 100 kD was applied for DNA purification and concentration:  Insert the Amicon Ultra-0.5 device into one of the provided microcentrifuge tubes.
 Add up to 100 μL of sample and 400μl pcr buffer with Mg2+ to the Amicon Ultra filter device.
 Spin the device at 4500 × g for 15 minutes and discard the liquid in the microcentrifuge tube.
 Repeat steps 2 and 3 for 4 times.
 Place the filter device upside down in the microcentrifuge tubes, spin for 4 minutes at 1000 x g to transfer the concentrated sample from the ultra filter device to the tube.

TEM Imaging
Transmission Electron Microscopy (TEM) was applied to image the DNA origami structures (tweezers B). In this part, an additional DNA staining step by 2% uranyl acetate solution was included.
 The plasma cleaned grids are clamped on with tweezers.
 5 µ L of sample is applied on each grid and then incubated for 5-10 minutes.
 The liquid is then removed from the grid using a filter paper.
 For highly concentrated samples: an additional washing step is included using 5 μL of ultrapure water.
 For samples containing DNA: 4 µ l of 2% uranyl acetate is applied and directly removed using a filter paper. Afterwards a second drop (4 µ l) of uranyl acetate was applied and let stay on the grid for 10 seconds before removing it again.
 The grids are dried for 20-30 minutes before they are stored in a grid sample holder.

AFM Imaging
The observation of the structure of nano-fingers was conducted under atomic force microscopy (AFM).
Coverslips and tapping mode in fluid were applied for sample preparation to observe the structure of nano-finger. The coverslip surface has to be completely flat in order to achieve best results.
 Coverslip is dipped in 0.1mg/mL poly-L-lysine (diluted with 1×PBS ) overnight and dried in air.
 Add 7 uL of sample to the surface and incubate for 10 minutes.
 After that, wash it with 100ul 1×TAE buffer twice and keep wet.

Fluorescence Detection
We monitor the real-time opening and closing of the tweezers by labeling the tweezer A arms with Cy3 and Cy5 fluorescent dyes, respectively. Cy3 exhibits lower fluorescence in the closed state since part of its energy transfers to Cy5, whereas Cy5 exhibites higher fluorescence under the same conditions. The gradual decrease in the intensity of Cy3 and Cy5 fluorescence observed over time can be attributed to photo bleaching.

FRET Fluorescence Imaging
Laser scanning confocal microscope(LSCM) is used to observe the real-time FRET.
 sample preparation sample 1: nano-fingers were incubated for 4 hours at 37°C.
sample 2: Add fuel into nano-fingers and incubate for 4h at 37°C; The samples are prepared on standard microscope slides to check for correct fluorescence labeling.
Then, the slide surface is coated with 0.1mg/mL poly-L-lysine (diluted with 1×PBS) which enables electrostatic binding of the negatively charged DNA as it creates a positively charged surface. The slide is dipped in the buffer overnight and dried in the air. 5 μL of specimen is directly deposited on the glass slide and incubated for 15 minutes at room temperature out of light and then covered with a piece of coverslip before mounting onto the sample stage.

 Fluorescence microscope observation
Fluorescence imaging is carried out on an LSCM in the sequential line mode and pictures are taken in a final imaging magnification of 100 fold. The donor dye Cy3 on nano-fingers is excited using the 550-nm laser. The resulting fluorescence, Cy3's and acceptor dye Cy5's emitted light, is recorded in two independent channels set up to detect emitted spectra correspondingly in the ranges 570-625 nm(for Cy3) and 655-755 nm(for Cy5). By this sequentially scanning the specimen with the individual laser(550 nm) and detecting fluorescence in two channels, Cy3's and Cy5's emitted light can be clearly exhibited on images which shows us whether FRET occurs. Images in each group are captured in the same region in two channels respectively. The laser beam alignment and imaging process are automatically controlled by the software.

Fluorescence spectrophotometry
FRET-recording was conducted by a Hitachi F4500 fluorescence spectrophotometer using a quartz cuvette at room temperature. The concentration of nano-fingers is 20 nM. Donor (Cy3) fluorescence is excited by illumination at 550 nm, and donor emission is measured at 565 nm. Acceptor (Cy5) is excited at 646nm, and acceptor emission is measured at 664 nm. Both the excitation slit and emission slit are 5 nm, and is measured at time scanning mode. After each addition of fuel or antifuel, the sample is mixed by rapid pipetting for about 30s. During this time, the fluorescence signal is collected continuously by the instrument, Each addition of fuel or antifuel represent a 20% stoichiometric excess over the previous addition.

Protein conjunction
Nano-fingers and thrombin were mixed at different concentrations and incubated for 2 hours at 25°C before gel electrophoresis. The concentration of nano-fingerss was hold at 20 nM and the concentration of thrombin is set as the ration 1:0, 1:2, 1:5, 1:10 and 1:20. Nano-fingers loaded in each lane is at equal amount. The gel containing 1.5% agarose and 0.1% ethidium bromide runs for 40 minutes at constant 80 V and is observed under UV.

Prothrombin time test
We take the clotting time in fibrinogen test as the qualitative analysis. 0.6 uL of nano-fingers/control and thrombin mixture was added to 300 uL bovine Fibrinogen with red ink in a 2 mL centrifuge tube. Samples were place a in 37°C water bath at 1 minute interval, totally 9 samples. After that, samples were taken out and inverted upside down on a flat platform. In this way, the number of the first tube with reactant did not fall down indicated the coagulation time.
3.6.2 Enzyme activity assay The enzymatic activity was measured by multimode Reader as quantitative analysis of coagulation reaction.
0.4 uL of nano-fingers/control was added into 200 μL bovine Fibrinogen with thrombin in 96-well plates at 37°C. During the measurement, the recording light wavelength was hold at 480 nm. The incubation time is 30 seconds, so is the testing time. The measurement stops while the absorbance value remains unchanged. Tests were condutcted for three times respectively to eliminate errors.