DNA multi-bit non-volatile memory and bit-shifting operations using addressable electrode arrays and electric field-induced hybridization

DNA has been employed to either store digital information or to perform parallel molecular computing. Relatively unexplored is the ability to combine DNA-based memory and logical operations in a single platform. Here, we show a DNA tri-level cell non-volatile memory system capable of parallel random-access writing of memory and bit shifting operations. A microchip with an array of individually addressable electrodes was employed to enable random access of the memory cells using electric fields. Three segments on a DNA template molecule were used to encode three data bits. Rapid writing of data bits was enabled by electric field-induced hybridization of fluorescently labeled complementary probes and the data bits were read by fluorescence imaging. We demonstrated the rapid parallel writing and reading of 8 (23) combinations of 3-bit memory data and bit shifting operations by electric field-induced strand displacement. Our system may find potential applications in DNA-based memory and computations.


Supplementary Figure 5. Computational simulations of electric field-induced distribution of ions and molecules. a,
A cross section of a portion of the chip used for electric field-induced hybridization. reff : effective radius of cell. Yellow/red strings: immobilized DNA molecules in a 10 m 2% agarose hydrogel layer. b, Electrical field and potential distribution. The electrical field distribution is indicated by the color contour lines while the potential distribution is indicated by the color map. Left color bar: color map for electric field strength contour lines (in V/cm). Right color bar: electric potential color map (in V). c,d, intensity along the vertical (c) and horizontal (d) green dashed lines in b. e-h, Spatial distribution of histidine molecules (e), Na + (f) and Cl -(g) ions, and DNA (h) under applied electric fields. The plots show the intensity profiles along the horizontal green dashed lines in the intensity heat maps. Scale bars are 80 µm. Figure 6. Current density profiles from FEM simulations. a, 2D plot of current density (A/cm 2 ) when a 1.5 VDC is applied to the central electrode while the other two adjacent electrodes were maintained at 0 V. b, 3D plot of current density. The current density is plotted along the vertical axis. The distance is plotted along the X-Y plane. Left color bar: color map for the electric field strength contour lines on the XY plane (in V/cm). Right color bar: color map for the current density along the Z axis (in A/cm 2 ). Scale bar is 80 µm.

Computational simulations of electric field-facilitated transport of ions and molecules
To gain a better understanding of the EFH process and to optimize the operations of our DNA NVM systems, we simulated the electric field strength and distribution, and the electric field-induced distributions of histidine molecules, Na + , Cland DNA molecules over the cells of a Nanogen microchip with an array of 100 individually addressable electrodes. The simulations were performed using the COMSOL Multiphysics 5.2 software package (COMSOL Inc.).
Supplementary Fig. 5a shows a cross-sectional structure of one cell and its vicinity on the microchip. The thickness of the platinum electrodes is 200 nm. The diameter of the exposed area of the ring electrode is 80 µm. Except the exposed area of the ring electrodes, the surface is covered with a 20 µm thick SiO2 insulator layer. The entire surface is covered underneath a 10 µm hydrogel layer. The dimensions were determined using SEM. 1 The modeling of the electric field intensity and distribution was performed using a conductivity of 2.2 mS/cm, which is the sum of the conductivity of a buffer containing 10 mM NaCl in 1x TBE (1.0 mS/cm) and that of 2% agarose hydrogel (1.2 mS/cm). 2, 3 We used the Nernst-Planck equation to simulate the distribution of DNA and exchanges of ions on the chip with 1.5 VDC applied to the center electrode. 4,5 The effect of surface charge on the gel was assumed to be negligible due to the low density of DNA and high applied electric field. 6 The distributions of voltage, electric field and current were simulated by setting the central electrode to 1.5 V and other electrodes to ground. The Nernst-Plank equation was used to calculate the distributions of ions and DNA. The electric mobility of the Na + and Clwas set to 1.28x10 9 m 2 /s and 1.77x10 9 m 2 /s, respectively, according to the literature. 7 The net charge of a 17-base long DNA molecule was assumed to be -17e.
The results from the simulations are shown in Supplementary Fig. 5b-f. Supplementary Fig. 5b, c show the distribution of the electric field and field lines, and the potential profiles in both the vertical and horizontal directions around the center electrode. As can be observed, the area with the highest field strength is around the center electrode with an effective area (with > 0.6 V) of about ~100 m, slightly greater than the physical size (80 m) of the electrode. The distribution of histidine molecules is shown in Supplementary Fig. 5d. Most histidine molecules are positioned near the center electrode with a profile slightly broader than those of simple inorganic ions, very likely due to its ability to change ionization state by the H + and OHions produced by the electrochemical hydrolysis of water. 8 This ability to neutralize the H + and OHions minimizes the electrochemical attack on other molecular species and the hydrogel layer. Supplementary Fig. 5e-f show the distributions of Na + and Clions.
Finally, the distribution of DNA molecules is shown in Supplementary Fig. 5g. A 17-base long ssDNA was used for the simulation and the effect of the gel matrix on the electrophoretic migration of the DNA was not considered. The ssDNA is concentrated at the positive electrodes after the application of the positive potential for only 90 s. In practice, the bit-encoding DNA molecules very likely hybridize to the encoding DNA template molecules already immobilized on the hydrogel surface and matrix upon encountering the gel layer during the migration toward the electrodes.
We also determined the Faradaic current distribution (Supplementary Fig. 6). The current mostly flows at the edges of the positive electrode. In our experiments, we found that the application of a high current could cause the detachment of the hydrogel layer from the surface due to electrochemical hydrolysis ( Supplementary Fig. 7).

Optimization of electrophoretic transport and electric field-induced hybridization
Repetitive writing, reading, and logical operations are required to operate the DNA MLC-NVM systems. Ideally, random-access activation, writing by EFH and bitwise manipulations by EFD of the memory cells can be performed in parallel in a relatively small number of steps, and the operations are carried out using reactive DNA components in a solution over the entire memory array. In addition to operation speed, robust and reproducible operations of the system should also be taken into consideration. Guided by computational simulations, we experimented with key parameters, which include the applied electric potential, concentrations of DNA and buffers, to identity the working conditions. For the particular 100-site Nanogen microchips with individually addressable ring electrodes (80 µm in diameter, 200 µm spacing) covered under a 10 µm thick 2% agarose hydrogel layer, we found that efficient EFH could be performed in a buffer containing 50 mM histidine, 10 mM NaCl and 20 µM of DNA by applying 1.5 V to the electrodes for 1.5 minutes without electrochemical damage to the hydrogel layer. Some typical results are shown in Supplementary Fig. 8.
We also investigated the specificity of random access to the memory cells by addressing only certain cells in localized areas of the 100-site microchips. As described in the main text, a biotinylated dA25 DNA template was used to encode the data bit. A Cy3-labeled dT25 was used to write the data bit. The writing and reading operations were performed as described in the main text. Supplementary Fig. S5 shows the fluorescence images of three sub-arrays of memory cells on the same microchip. The subarrays were written and read one at a time.