Stacking-induced fluorescence increase reveals allosteric interactions through DNA

Abstract

From gene expression to nanotechnology, understanding and controlling DNA requires a detailed knowledge of its higher order structure and dynamics. Here we take advantage of the environment-sensitive photoisomerization of cyanine dyes to probe local and global changes in DNA structure. We report that a covalently attached Cy3 dye undergoes strong enhancement of fluorescence intensity and lifetime when stacked in a nick, gap or overhang region in duplex DNA. This is used to probe hybridization dynamics of a DNA hairpin down to the single-molecule level. We also show that varying the position of a single abasic site up to 20 base pairs away modulates the dye–DNA interaction, indicative of through-backbone allosteric interactions. The phenomenon of stacking-induced fluorescence increase (SIFI) should find widespread use in the study of the structure, dynamics and reactivity of nucleic acids.

Metallation‐Induced Heterogeneous Dynamics of DNA Revealed by Single‐Molecule FRET

“…For the donor-only hairpin, the interaction with Os1ÀCl prevents closing and thus stacking, resulting in ad ecreasei nC y3 emission intensity as salt is increased (Supporting Information, Ta ble S6 and Figure S5). [20] The other difference between experiments with Cy3 and Cy3B is that Cy3 displays three lifetime components, which are attributedt od ifferentl ocal dye environments,asd iscussed previously. [20] To investigate whethert he Os1ÀCl is targeting the GC-rich stem, an ew hairpin (H4-Cy3) was designed with an AT-rich stem and similart hermodynamic stabilitya st he GC-rich hairpin (Supporting Information, Figure S6).…”

“…[20] The other difference between experiments with Cy3 and Cy3B is that Cy3 displays three lifetime components, which are attributedt od ifferentl ocal dye environments,asd iscussed previously. [20] To investigate whethert he Os1ÀCl is targeting the GC-rich stem, an ew hairpin (H4-Cy3) was designed with an AT-rich stem and similart hermodynamic stabilitya st he GC-rich hairpin (Supporting Information, Figure S6). In the absence of the Os1ÀCl,t he addition of NaCl resulted in ad ecrease of the steady-state intensity of the Cy3 emission and ac orresponding increasei nt he emission from Cy5 (Supporting Information, Figure S7 and Table S7).…”

“…In addition to changes in the extent of energy transfer for the different donord yes (e.g.,d ue to Fçrster radius, dye-dye distance), we previously observedt hat Cy3 intensity increases upon hairpin closing due to stacking in the gap. [20] This was termeds tacking-induced fluorescence increase (SIFI) and is due to the prevention of photoisomerisation upon stacking. For the donor-only hairpin, the interaction with Os1ÀCl prevents closing and thus stacking, resulting in ad ecreasei nC y3 emission intensity as salt is increased (Supporting Information, Ta ble S6 and Figure S5).…”

Probing DNA Dynamics: Stacking‐Induced Fluorescence Increase (SIFI) versus FRET

“…These requirements can limit the applicability of the method. In contrast, we recently demonstrated that both local and long‐range information on DNA structure and dynamics could be obtained using only a single fluorescent dye . We showed that the fluorescent dye Cy3 is able to stack site‐selectively in DNA with a resultant enhancement in fluorescence, which we termed stacking‐induced fluorescence increase (SIFI) …”

“…SIFI also works via modulation of photoisomerisation, and can be considered as a subset of NAIFE, exploiting the specific interaction between Cy3 and a nick or gap in the double‐stranded DNA (dsDNA) structure. We previously used SIFI to detect hybridisation of a DNA hairpin at the single‐molecule level, and to sense a long‐range perturbation in the form of an abasic site up to 20 base pairs (bp) from the site of dye stacking . Here we directly compare SIFI and FRET (using Cy3 as the donor and Cy5 as the acceptor) at the single‐molecule level (termed smSIFI and smFRET, respectively) as tools for probing the dynamics of DNA.…”