Conformational flexibility in DNA nanoconstructs: A time-resolved fluorescence resonance energy transfer study

Time-resolved fluorescence resonance energy transfer has been used to investigate the conformational flexibility of a DNA nanostructure. As the size of DNA nanostructures are becoming increasingly smaller, commonly used methods for characterization like atomic force microscopy (AFM) will become increasingly more difficult. Here we demonstrate that time-resolved fluorescence resonance energy transfer (tr-FRET) can serve as a tool for retrieving structural information on small DNA nanostructures. We have formed a DNA pseudohexagon using six different standard 22-mer oligonucleotides. The construct was labeled with a donor (fluorescein) in one corner while "walking" the acceptor (Cy3) from corner to corner around the hexagon. This resulted in five differently labeled constructs that were investigated with time-correlated single photon counting. The distribution of donor-acceptor distances present in each sample was estimated from analyzing the donor emission decays using a Gaussian distribution model. The results show a relatively wide distribution for all measured distances, fwhm between 14 and 33 angstrom, indicating that the pseudohexagonal motif is a flexible structure. In addition the recovered mean distances between the donor and acceptor follow the expected theoretical trend very well. The results show that, not only is tr-FRET ail alternative method for retrieving structural information from DNA nanostructures whose size are below the present resolution of AFM but also, it demonstrate the potential of tr-FRET as a method for probing local flexibility in larger arrays of this kind.