2-Aminopurine Single-Molecule Fluorescence

Abstract

Base flipping is a local conformational change that results in the rotation of a nucleotide out of its helical structure. This is frequently observed in numerous nucleic acid structural rearrangements, such as in DNA-protein interactions and in different RNA enzymes. 2-Aminopurine (2AP) is a fluorescent nucleotide analog extensively used to probe local conformational changes in nucleic acids in bulk experiments, but no single molecule approaches have been developed to study these mechanisms using 2AP. One challenge towards this goal is that 2AP fluorescence is very sensitive to the interaction with neighboring nucleotides, and its emission spectrum partially overlaps with that of tryptophan. We have developed new click-chemistry-based surface immobilization approach that enables us to monitor, in real time base flipping in DNA. We have characterized the fluorescence properties of single 2AP labelled DNAs. Our results show that nucleotides are very dynamic in a single- or double-stranded DNA and that bases can flip out of the helix, suggesting that the base flipping occurs frequently and in a much slower time scale that previously believe. This new assay can also be used to study other local conformational changes in nucleic acids at the single-molecule level by using 2AP as the base flipping probe.