Complete Mapping of DNA‐Protein Interactions at the Single‐Molecule Level

Abstract DNA–protein interaction plays an essential role in the storage, expression, and regulation of genetic information. A 1D/3D facilitated diffusion mechanism has been proposed to explain the extraordinarily rapid rate of DNA‐binding protein (DBP) searching for cognate sequence along DNA and further studied by single‐molecule experiments. However, direct observation of the detailed chronological protein searching image is still a formidable challenge. Here, for the first time, a single‐molecule electrical monitoring technique is utilized to realize label‐free detection of the DBP–DNA interaction process based on high‐gain silicon nanowire field‐effect transistors (SiNW FETs). The whole binding process of WRKY domain and DNA has been visualized with high sensitivity and single‐base resolution. Impressively, the swinging of hydrogen bonds between amino acid residues and bases in DNA induce the dynamic collective motion of DBP–DNA. This in situ, label‐free electrical detection platform provides a practical experimental methodology for dynamic studies of various biomolecules.

. DNA sequence information Table S2. Kinetic and thermodynamic analysis of concentration-dependent experiments S1. Protein purification and characterization Figure S1. Strategical demonstration of single-molecule experiments of the DNA-DBP system. A single-molecule DNA biosensor is constructed on silicon nanowire field-effect transistor (FET) devices and the electrical signals are measured and recorded so as to realize real-time monitoring of the protein-DNA binding process with high time resolution.
Details of protein expression/purification and Isothermal Titration Calorimetry (ITC) Assays are provided in the Experimental Section of the main text. The ITC results [1] (Figure S2, adopted from Ref. 1) showed that the binding ability of DNA to WRKY1N decreased by 1-2 orders of magnitude due to the mutation of the recognition sequence (GGTC). The relative position of GGTC in DNA had little effect on the binding ability. K122 mutation of WRKY1N also led to a significant decrease in the binding ability. Theoretical pI: 9.20.
All structure figures were generated by PyMOL. According to Figure S3, GGTC is the core sequence recognized by WRKY1N protein. Y119 and K122 residues interact directly with G and C in the sequence.   [1] . a-c) The crystal structure of a WRKY1N-DNA complex (adopted from Ref. 34, PDB code: 6J4E) at different angles shows the hydrogen bonds between protein residues and DNA (a, specific bases; b and c, phosphate skeleton). d) Schematic diagram of the interaction between protein residues (green) and DNA. Yellow arrows represent hydrogen bonds between residues and bases. Blue arrows represent hydrogen bonds between residues and phosphate skeleton. Protein residues and GGTC sequence have the strong interaction.  SiNW growth Procedure: The nanowire growth procedure is similar to those reported in the previous studies [2,3] . Gold nanoparticles (AuNPs, Sigma-Aldrich, the average diameter of ~20 nm) were used as catalysts dispersing on silicon wafers with a 300 nm thick thermal oxide layer.

Surface modification of SiNWs:
We carried out an in-situ modification of SiNWs. The wafer on which we grew the silicon nanowires and 10-20 μL triethoxy (3-succinate propyl) silane (TESPSA, 95%, J&K, in a 1.5 mL microtube) were placed in a vacuum desiccator, which was then heated at 120 o C for 2 hours. This operation also remains the original growth morphology of SiNWs and enables the further transfer of functionalized SiNWs [4,5] .
SiNW transfer and FET fabrication: After vapor modification, the functionalized SiNWs were transferred to a 1.4 cm × 1.8 cm silicon substrate with a 1000 nm thick thermal oxide layer and well-aligned by mechano-sliding [5,6] . The electrode patterns were defined by a standard UV lithography (BG-401A, China electronics technology Group Corporation). After the etching of SiNWs with a buffered HF solution (40% NH4F:40% HF, 7:1) to remove the oxide shell, 8 nm Cr and 80 nm Au were deposited through thermal evaporation (ZHD-300, Beijing Technol Science) to form metal electrodes. A 30 nm-thick SiO2 protective layer was then deposited through electron beam thermal evaporation (TEMD-600, Beijing Technol Science) in order to passivate the contact interface. After lift-off with acetone, the surface-modified SiNW FET devices were obtained ( Figure S6).  Figure S7).

Single-molecule protein decoration:
The procedure is similar to those reported in previous studies [3,7] . A gap-opening procedure was carried out to decorate the SiNW-FET device with single DNA molecule. After the device fabrication, a PMMA layer (950, A4) was spin-coated (4000 rpm, 45 s) on the surface and then baked at 180 °C for 2 min. The high-resolution electron beam lithography (EBL) was then applied to introduce a design line pattern with a ~5 nm-wide at the specific position to obtain the window precursor ( Figure S5                Data representation mean ± SD, sample size n = 3.