A polarized view on DNA under tension

Abstract

In the past decades, sensitive fluorescence microscopy techniques have contributed significantly to our understanding of the dynamics of DNA. The specific labeling of DNA using intercalating dyes has allowed for quantitative measurement of the thermal fluctuations the polymers undergo. On the other hand, recent advances in single-molecule manipulation techniques have unraveled the mechanical and elastic properties of this intricate polymer. Here, we have combined these two approaches to study the conformational dynamics of DNA under a wide range of tensions. Using polarized fluorescence microscopy in conjunction with optical-tweezers-based manipulation of YOYO-intercalated DNA, we controllably align the YOYO dyes using DNA tension, enabling us to disentangle the rapid dynamics of the dyes from that of the DNA itself. With unprecedented control of the DNA alignment, we resolve an inconsistency in reports about the tilted orientation of intercalated dyes. We find that intercalated dyes are on average oriented perpendicular to the long axis of the DNA, yet undergo fast dynamics on the time scale of absorption and fluorescence emission. In the overstretching transition of doublestranded DNA, we do not observe changes in orientation or orientational dynamics of the dyes. Only beyond the overstretching transition, a considerable depolarization is observed, presumably caused by an average tilting of the DNA base pairs. Our combined approach thus contributes to the elucidation of unique features of the molecular dynamics of DNA.

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

“…1B). We observe that both before and within the OST (regimes 1 and 2, respectively), fluorescence emission remains negatively polarized, and dyes are efficiently excited only using an excitation polarization perpendicular to the DNA axis ( 21 , 24 ). This indicates that intercalators align parallel to the DNA base pairs and perpendicular to the DNA axis (Fig.…”

“…Our initial experiments quantify the polarization response of the cyanine intercalator YOYO-1 ( 30 ) at different DNA extensions ( 21 , 24 ). Following previous work ( 24 ), in which changes in fluorescence polarization were first observed at extensions beyond the OST, DNA was extended in the presence of intercalators using a low-salt imaging buffer (see Materials and Methods). This buffer (where intercalator unbinding is much slower than the stretching speed) ensures a relatively high dye coverage that persists even during DNA overstretching ( 16 ).…”

“…1D has been reported before by van Mameren et al . ( 24 ), who proposed two hypotheses to explain this unexpected change: (i) The orientation of an intercalator within its binding site may undergo rapid rotational diffusion in the time interval between excitation and fluorescence emission. Such random reorientations or “wobble” may occur while the mean axial tilt with respect to the DNA axis (θ) remains close to 90°.…”

“…Along these lines, van Mameren et al . ( 24 ) used a combined excitation/emission polarization technique to measure the response of intercalated dyes when DNA was stretched to different extensions using optical tweezers. That work revealed pronounced changes in fluorescence polarization as DNA was extended beyond the OST.…”

Bio-Molecular Applications of Recent Developments in Optical Tweezers

“…A similar dual-trap plus microfluidics plus widefield fluorescence (both TIRF and epi-illumination) has been used by another group to study RecA, another protein involved in DNA repairing [98]. The integration of high-resolution OT with polarized fluorescence microscopy has been demonstrated in a very recent study [99] of the behavior of YOYO-intercalated double-stranded DNA (dsDNA) subject to forces up to 80 pN, well beyond the DNA overstretching transition. The use of linearly polarized excitation light allowed to check the orientation of the dyes, perpendicular to the axis of the DNA, and to characterize the fast dynamics that they undertake in the time between excitation and emission.…”