Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA

Abstract

DNA structural transitions facilitate genomic processes, mediate drug-DNA interactions, and inform the development of emerging DNA-based biotechnology such as programmable materials and DNA origami. While some features of DNA conformational changes are well characterized, fundamental information such as the orientations of the DNA base pairs is unknown. Here, we use concurrent fluorescence polarization imaging and DNA manipulation experiments to probe the structure of S-DNA, an elusive, elongated conformation that can be accessed by mechanical overstretching. To this end, we directly quantify the orientations and rotational dynamics of fluorescent DNA-intercalated dyes. At extensions beyond the DNA overstretching transition, intercalators adopt a tilted (θ ~ 54°) orientation relative to the DNA axis, distinct from the nearly perpendicular orientation (θ ~ 90°) normally assumed at lower extensions. These results provide the first experimental evidence that S-DNA has substantially inclined base pairs relative to those of the standard (Watson-Crick) B-DNA conformation.

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

“…This curve ( Figure 3J) matches well with the DNA hyperstretching curve measured by optical tweezer experiments in the presence of the fast equilibrating dye YO-PRO-1 ( Fig. S1 in reference 56 ). Both curves feature a slow increase in extension in the low-force regime, a rapid increase until ~100 pN, and a slow increase above 100pN.…”

“…Over the course of the experiment, the total intensity of the entire DNA molecule showed slow dissociation of the YOYO-1 dye but no clear changes correlated with changes in the length of the DNA (Figure 3C, Supplementary Figure 2). This suggests that the YOYO-1 did not dissociate, the intensity of each YOYO-1 did not change, and the dsDNA did not peel during the experiment, consistent with a direct transition into the HS state 49,56 . Therefore, the change in intensity profile is mostly due to the change in dye distribution that results from local stretching of the molecule.…”

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

“…[9][10][11][12][13] Even with multiple biotin-streptavidin bonds, the maximum force reported for near equilibrium conditions on a time scale of 1-10 minutes is ∼ 180 pN. 14,15 In some cases, parts of the molecule can strongly adsorb onto gold surfaces and AFM tips, allowing constant application of hundreds of pN. 16 When physical adsorption is unavailable or undesirable, more involved covalent conjugation chemistries are needed for high force application.…”

Supercoiling DNA optically

“…To this end, we envision that the technique could be even further functionalized through integration with, for example, FRET, 24 super-resolution imaging 23 and fluorescence polarization microscopy. 30…”