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 double-stranded 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.
Highlights
With the advent of single-molecule observation techniques, DNA has become a model system for studying the dynamics of semi-flexible polymers.1–4 The direct visualization of fluorescently labeled DNA undergoing Brownian motion using DNA-specific fluorescent dyes has allowed for a quantitative understanding of its thermal fluctuations.5–7 Many dyes specific to double-stranded DNA have been employed in fluorescence applications both in bulk and at the microscopic level
Force-extension analysis was used to reveal whether a single DNA molecule was captured, upon which the DNA was moved to a buffer containing 100–200 nM YOYO
We have demonstrated the application of single-molecule manipulation techniques to polarized fluorescence microscopy of individual DNA molecules
Summary
With the advent of single-molecule observation techniques, DNA has become a model system for studying the dynamics of semi-flexible polymers. The direct visualization of fluorescently labeled DNA undergoing Brownian motion using DNA-specific fluorescent dyes has allowed for a quantitative understanding of its thermal fluctuations. Many dyes specific to double-stranded DNA (dsDNA) have been employed in fluorescence applications both in bulk and at the microscopic level. Many dyes specific to double-stranded DNA (dsDNA) have been employed in fluorescence applications both in bulk and at the microscopic level These dyes can be classified according to their mode of binding to DNA into two major categories: groove binding dyes (e.g., DAPI or Hoechst, binding in the minor groove) and intercalating dyes (e.g., ethidium bromide, SYBR dyes, or cyanine dyes such as TOTO, YO, and YOYO). The latter are planar organic structures that bind dsDNA by sandwiching in between consecutive base pairs [Fig. 1(a)], thereby lengthening the DNA upon binding.. The absorption and emission dipole moments are roughly perpendicular to the DNA helix axis.
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