Flexibility of single-stranded DNA measured by single-molecule FRET

The mechanical flexibility of ssDNA is crucial for understanding the biological machinery but its characterization has been difficult due to the lack of an experimental tool that measures the structure of ssDNA in the nanometer scale. Here, we demonstrate that single-molecule FRET can be used to probe the structures of a flexible ssDNA. We designed a dsDNA with various lengths of single-stranded overhang and determined the flexibility of the single-stranded segment by measuring the FRET value. We found that three of our ssDNAs with lengths shorter than the persistence length are indeed long enough to undergo folding. Since metal ions present in solution can affect the flexibility of DNA, we employed Na+ and Mg2+ at different concentrations. We found that there is no significant effect of charge screening by metal ion when the ssDNA is less than 9 bases in length but it becomes appreciable for longer ssDNAs. [Display omitted] •We investigated the flexibility of ssDNA using a single-molecule technique.•ssDNA even shorter than the persistence length was found to be flexible.•Divalent cation is effective in screening the negative charge of the ssDNA.