Oriented Soft DNA Curtains for Single-Molecule Imaging

Over the past 20 years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes a flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using a protein template-directed assembly. In our assay, a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein–DNA interactions at more controllable reaction conditions. Additionally, we increased the immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g., CRISPR-Cas9) binding assay that monitors the binding location and position of individual fluorescently labeled proteins on DNA.

Portable printing device (PPD). The basic principles of our PPD device ( Figure S2) are similar to the previously published one, 1 and it was assembled from commonly used parts in an optics laboratory. As the Figure S2A shows, firstly we placed the Si master on a silicone rubber sheet attached to a glass slide (microscope slide, 1 mm thickness, Heinz Herenz Hamburg), which was glued to a base plate (3BP4-02, Standa) using a piece of double sided sticky tape (9088-200, 3M).
We made holes in the centers of the silicone rubber sheet, the glass slide, and the double-sided sticky tape. To keep the Si master fixed on the silicone rubber sheet, we sucked out the air through this hole. For this purpose, we glued a nanoport (10-32, IDEX Health & Science) to the center of the under side of the glass slide and connected it to a 2 mL syringe (syringe 1 in the Figure S2A, Omnifix, LY21.1 Carl-Roth) via a 30 cm long tubing (a suitable tubing clamp was installed to clamp the tube after the air was sucked out). After this step, we attached a protein-coated PDMS elastomer with a protein ink monolayer flat-side-up onto the glass slide using double-sided sticky tape ( Figure S2B). Next, we held the glass slide with tweezer and positioned the PDMS elastomer facing inked-side-down onto the Si master. To apply printing pressure, we made a system of two tubing-connected syringes ( Figure S2C, 1 mL Omnifix-F Solo Single-use syringe, 9161406V, Braun). First, we fitted the first syringe (syringe 2 in the Figure S2C) into the center hole of two connected base plates (3BP4-02, Standa). Second, we filled the syringes and the tubing with tap water and fixed the syringes to the 15 cm long tubing (228-0703, VWR) using tube clamps (EX52.1, Carl-Roth) and 3 cm long adaptor tubings (N874.1, Carl-Roth). These adaptor tubings were placed on the ends of the 15 cm long tubing and made the contact with the syringe stronger.
Once the system was assembled, we mounted it onto mounting posts (3MP-25+3AH6-4, Standa) using suitable screws (M6, 3 cm long). Finally, we applied the printing pressure to the PDMS elastomer using the second syringe (syringe 3 in the Figure S2C) and controlled the printing pressure by visually inspecting the volume scale on the second syringe.
Printing force measurements. To measure the actual printing force applied with the device, we employed a force sensitive resistor (FSR 400 Series Round, 5.08 mm 2 active area, 0.35 mm nominal thickness, Interlink Electronics, Inc.), which was placed in between syringe 2 and the glass slide holding the PDMS elastomer ( Figure S2C). For this measurement we followed previously described procedure. 2,3 One leg of the FSR device was connected to the +5 V (V in ) output port of the Arduino Uno card and the other leg was tied to a measuring resistor (R m = 10 kΩ) in a voltage divider configuration (i.e. one end was connected to ground and the other to the analog input port of the Arduino Uno card). The output voltage (V out ), which was monitored using the Arduino Uno card, increased with increasing force. To calculate the actual printing pressure in Newtons per square centimeter (N), we used the following equations for resistance (R) and conductance (C). Note, our PDMS elastomer had 5 x 5 mm 2 dimensions, therefore to get the force in N/cm 2 we introduced multiplication factor 4 into equation 3 (multiplication by the factor of 4 is embedded in the equation). Finally, we used conversion factor (k) obtained from the calibration curve of this sensor to calculate the actual force. 3 The obtained syringe position -printing pressure calibration curve is shown below in Figure S3.

Functionality of traptavidin.
We made sure that our tAv was indeed functional and bound biotinylated 5 kb long DNA molecules. For that purpose we acquired TIRF images at 488 nm wavelength excitation and they showed no DNA binding on the surface modified by only m-PEG ( Figure S7A), while the m-PEG/bt-PEG mixture modified surface was densely covered with DNA molecules ( Figure S7B). Both surfaces were incubated with 0.02 mg/mL tAv, washed, incubated with 5 kb long DNA, washed and then stained with SG. These results verified functionality of the surface bound tAv molecules.

Functionality of biotinylated antibodies directed against digoxigenin.
We prepared biotinylated anti-dig antibodies by conjugating them with Biotin-PEG4-NHS ester. Functionality of these bt- anti-dig conjugates was tested on a surface. The bt-anti-dig were immobilized on a PEGylated surface (10% bt-PEG) covered with sAv. Next, the singe-end dig-labeled λ DNA (dig-λ DNA) molecules were immobilized, stained using SG, and visualized using TIRF microscopy at 488 nm excitation without ( Figure S10A) or with buffer flow ( Figure S10B). These images showed mainly one-end tethered DNA fragments, suggesting successful anti-dig and dig-λDNA interaction. No binding of dig-λ DNA was observed on the PEGylated surface (10% bt-PEG) covered with only sAv.