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.
Highlights
We have previously developed a method that combines classical MD simulations with quantum mechanical/molecular mechanical (QM/MM) [33, 34] calculations to simulate fluorescence spectra with and without fluorescence resonance energy transfer (FRET) quenching. [35, 36] This approach was used to shed light on the important question of how conformational ensembles determine spectroscopic features, and it was shown that the common isotropic assumption made in many FRET studies must be corrected using simulation data
We show how important quantities that describe FRET quenching can be calculated from the simulation data using a QM/MM approach, and we discuss these for different relative chromophore geometries
We use the conformational ensemble of system 1 to calculate steady-state absorption and emission spectra, and to calculate FRET between the chromophores in this system
Summary
In contrast to the simulation at 310 K, the three terminal base pairs at the terminus at which PPa is attached are more disrupted at 363 K, which can be seen from the analysis of the Watson-Crick hydrogen bonds (Figure P in S1 File) and from visual inspection (Fig 5). [85] In systems 1 and 2, which resemble “complete” nucleic acid detection systems, the potential FRET donor (fluorescein) and the potential acceptor (PPa) tend to form close contacts in the simulations (when starting from a separated geometry).
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