DNA crossover flexibilities upon discrete spacers revealed by single-molecule FRET

In this study, we utilized the origami technique to integrate various types of spacers into the double-stranded crossover and examined their flexibilities using single-molecule fluorescence resonance energy transfer (smFRET). We discovered that for the traditional Holliday Junction connection with zero-base spacers, the inter-structural angle measures 58.7 degrees, which aligns well with previous crystallographic research. When introducing non-complementary double-stranded spacers as a free leash, we observed that longer spacers resulted in a more relaxed connection. In contrast, when using complementary segments, the two origami structures rotated as the number of base pairs increased, reflecting the structural characteristics of the B-duplex. Our findings indicate that a stable intramolecular duplex requires a minimum of 5 base pairs. Overall, our results highlight the potential for re-engineering crossovers and designing materials that can change volume with shrink-swell capabilities, as well as applications in torque sensing using short DNA duplexes.