Single-Stranded DNA Curtains for Real-Time Single-Molecule Visualization of Protein−Nucleic Acid Interactions

Abstract

Single-molecule techniques have greatly advanced our understanding of many types of biochemical reactions. We have established strategies for anchoring and organizing ‘double-stranded’ DNA (dsDNA) molecules on the surfaces of microfluidic sample chambers that are coated with a fluid lipid bilayer. This technique called “DNA Curtains” has proved powerful in the examination of proteins as they bind, diffuse and translocate along dsDNA. Single-stranded DNA (ssDNA) is a crucial intermediate in nearly all biochemical reactions related to the maintenance of genome integrity. However, most single molecule studies of proteins on ssDNA use short synthetic oligonucleotide substrates. Here, we present procedures for generating, aligning and visualizing hundreds of long single-stranded DNA molecules along the leading edges of nanofabricated barriers, where the DNA can either be “single-tethered” or “double-tethered”. This new approach permits long-desired access to critical biological reactions involving single-stranded DNA binding proteins.