DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes

Abstract

Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye via fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries.

Fig 1. Detecting specific nucleic acid sequences in a templated reacion.

F: 5-carboxyfluorescein, PS: photosensitizer (pyropheophorbide-a, PPa), L: linker cleavable by ¹O₂. [, –27]

Fig 2. 1–3: Structures of DNA-dye conjugates (green: PPa, orange: fluorescein) with linkers (magenta) cleavable by ¹O₂.

LA, LB: linkers: 1,9-dialkoxyanthracene (left) and 9-alkoxyanthracene derivatives (right).

Fig 3. DNA-dye conjugates with terminally attached dyes.

Fig 4. Structures of systems 1 (left) and 2 (right) at 0.51 ns (after equilibration with harmonic restraints, above) and at 230.5 ns (below, dyes and linkers shown as sticks, DNA as lines).

Ions and water were omitted for clarity. Figures were created using VMD. [76]

Fig 5. Structures of system 4 at 0.51 ns (after equilibration with harmonic restraints, above) and at 260.5 ns (below, left) and 220.5 ns (below, right), respectively. Left: Simulation at 310 K, right: simulation at 363 K.

Ions and water were omitted for clarity. Figures were created using VMD. [76]

Fig 6. Structure of system 5 at 0.51 ns (after equilibration with harmonic restraints, above) and at 220.5 ns (below).

Ions and water were omitted for clarity. Figures were created using VMD. [76]

Fig 7. Experimental (black) and calculated (red) steady-state absorption (left) and emission spectra of fluorescein, using a subset of the MD ensemble (424 snapshots) and the QM/MM methodology described in reference [35].

Additionally, the fitted ensemble of transition dipoles describing the S₁ → S₀ transition (emission spectrum) is shown (transition dipoles multiplied by a factor of 10). Note that the experimental spectra were recorded for systems with the chromophores attached to DNA and therefore additionally contain typical DNA absorption peaks in the UV region, while the calculated spectra resemble the situation of the chromophores in solution (due to the QM/MM formalism).

Fig 8. Experimental (black) and calculated (red) steady-state absorption (left) and emission spectra of PPa, using a subset of the MD ensemble (424 snapshots) and the QM/MM methodology described in reference [35].

Additionally, fitted ensembles of transition dipoles describing the S₀ → S_i transition with maximal oscillator strength (absorption spectrum) and the S₁ → S₀ transitions (emission spectrum) are shown (transition dipoles multiplied by a factor of 10). Note that the experimental spectra were recorded for systems with the chromophores attached to DNA and therefore additionally contain typical DNA absorption peaks in the UV region, while the calculated spectra resemble the situation of the chromophores in solution (due to the QM/MM formalism).