A new method for the covalent attachment of DNA to a surface for single-molecule studies

• Published in: Colloids and surfaces, B, Biointerfaces B, Biointerfaces, 2011-03, Vol.83 (1), p.91-95
• Main Authors: Schlingman, Daniel J., Mack, Andrew H., Mochrie, Simon G.J., Regan, Lynne
• Format: Article
• Language: English
• Subjects: Attachment; Beads; Binding; Biotin - chemistry; colloids; Covalence; Covalent bond; Covalent bonds; Deoxyribonucleic acid; DNA; DNA - chemistry; glass; methodology; Nucleotides; Optical Tweezers; Polyethylene Glycols - chemistry; Polystyrene resins; polystyrenes; researchers; Silane-PEG-NHS; Silanes - chemistry; Single-molecule; Streptavidin - chemistry; Succinimides - chemistry; Surface attachment; surface interactions; Surface Properties; Time Factors
• ISSN: 0927-7765; 1873-4367
• DOI: 10.1016/j.colsurfb.2010.11.002

[Display omitted] ▶ New method to covalently attach DNA to a surface using silane–PEG–NHS. ▶ The method creates a PEG monolayer to decrease non-specific adsorption. ▶ Method withstands higher pulling forces for longer times. ▶ Can pull DNA with an optical trap through its overstretching transition many times. Attachments between DNA and a surface or bead are often necessary for single-molecule studies of DNA and DNA–protein interactions. In single-molecule mechanical studies using optical or magnetic tweezers, such attachments must be able to withstand the applied forces. Here we present a new method for covalently attaching DNA to a glass surface, which uses N-hydroxysuccinimide (NHS) modified PEG that is suitable for high-force single-molecule mechanical studies. A glass surface is coated with silane-PEG-NHS and DNA is covalently linked through a reaction between the NHS group and an amine modified nucleotide that has been incorporated into the DNA. After DNA attachment, non-reacted NHS groups are hydrolyzed leaving a PEG-covered surface which has the added benefit of reducing non-specific surface interactions. This method permits specific binding of the DNA to the surface through a covalent bond. At the DNA end not attached to the surface, we attach a streptavidin-coated polystyrene bead and measure force-versus-extension using an optical trap. We show that our method allows a tethered DNA molecule to be pulled through its overstretching transition (>60pN) multiple times. We anticipate this simple yet powerful method will be useful for many researchers.