Visualizing Single DNA Molecules in Nanofluidic Channels

Abstract

Nanofluidic channels have become an important tool for studies of single DNA molecules. Studies have been done on DNA polymer physics as well as DNA mapping and DNA/protein interactions. Common for these studies is that the DNA is homogeneously stained, typically with the bis-intercalating dye YOYO. While this is beneficial for some studies, there are several applications where the dyes interfere with the study. YOYO changes physical properties, such as charge, persistence length, contour length and winding of the DNA, as well as causes DNA damage by photonicking when exposed to light. Furthermore, for studies of interactions between DNA and ligands or proteins intercalating YOYO competes with the molecule of interest for the available binding sites.In order to avoid these obstacles we have designed a 50 kbp DNA construct with quantum dots (QDs) in each end. This construct allows us to study single DNA molecules in nanochannels without any intercalated dyes, by measuring the distance between the two fluorescent ends. We demonstrate the effect YOYO-labelling has on DNA polymer physics and show how this novel DNA construct can be used for DNA/ligand interaction studies on the single molecule level.Furthermore, nanostructures have extreme surface-to-volume ratios, leading to that the negatively charged surfaces are sticky to most hydrophobic and/or positively charged molecules and particles, including the streptavidin coated QDs used for end-labelling. We therefore developed a passivation-scheme, based on PEG, that is resistant to the QDs sticking. The absence of QDs sticking to passivated nanochannels serves as a general demonstration that can be extended to most proteins, allowing us to do DNA/protein interaction studies in the nanochannels using end-labeled DNA.Acknowledgements:This project is funded by the Swedish Foundation for Strategic Research and the Chalmers Area of Advance in Nanoscience and Nanotechnology.