Fixed DNA Molecule Arrays for High-Throughput Single DNA–Protein Interaction Studies

Abstract

The DNA Curtains assay is a recently developed experimental platform for protein−DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA−protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single-and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single-and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA−protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

Oriented Soft DNA Curtains for Single Molecule Imaging

“…Si masters were fabricated and characterized according to the previously published procedure. 10 Fabrication of the Si master involves formation of a self-assembled monolayer (SAM) from 1-eicosanethiol (HS-C 20 , AlfaAesar), 16 surface patterning by the nanoshaving lithography technique using an AFM (NanoWizard3, JPK Instruments AG, Germany) and wet chemical etching. [17][18][19] Characterization of the Si master was performed using an upright optical microscope BX51 (Olympus, Japan) and the AFM, operating in AC mode.…”

“…Flat PDMS elastomer stamps for protein lift-off µCP were fabricated according to the previously published procedure. 10 Briefly, the prepolymer and curing agent (10:1 ratio w/w, Sylgard 184 kit) were thoroughly mixed, degassed in a vacuum desiccator (30 min), poured into a plastic Petri dish and cured in an oven (65 °C for 14 h). The thickness of the cast PDMS elastomer was ~2 mm.…”

“…The lift-off µCP was performed similarly to the published procedure. 10,20 The PDMS elastomer (5 x 5 mm 2 dimensions) was immersed in isopropanol for 10 min, held by tweezers and dried for 15 s, placed on a plastic Petri dish and dried for another 10 min. The Si master was immersed in isopropanol (20 min), held by tweezers and dried, and cleaned by air plasma (5 min, ~500 mTorr, high-power mode, PDC-002, Harrick, USA).…”

“…However, the original DNA Curtains are less stable and more expensive fabrication-wise than the platform described in this and our previous work. 10 The skybridge platform contains stably immobilized DNA molecules, but it utilizes rather unusual phenomena for visualization of fluorescently labeled DNA and proteins. However, it is an interesting alternative to the existing DNA Curtains platform.…”

“…Recently we demonstrated that streptavidin (sAv) patterns on the modified coverslip surface can be utilized to fabricate biotinylated DNA arrays. 10 The design of the protein patterns on the modified surface ensures predefined distribution and aligning of the biotinylated DNA molecules on the narrow line-features (> 200 nm). We refer to these aligned molecules as Soft DNA Curtains.…”