Photocages are powerful tools for spatiotemporal control of molecule release or biological activity. However, many photocages are unsuitable for biological experiments since they are mostly activated by harmful ultraviolet (UV) light and often lack a sufficient optical readout. Thus, there is a high demand for near infrared (NIR) and/or two-photon activatable photocages with a characteristic readout. In this report, we will study a supramolecular, covalently linked energy-transfer dyad based on a BASHY fluorophore serving as a two-photon antenna for a poorly two-photon absorbing BODIPY photocage. The herein investigated systems, with and without a leaving group (LG), show different excitation energy transfer (EET) efficiencies and therefore differ in their fluorescence properties. To understand the molecular basis for these significant differences, detailed spectroscopic and theoretical analyses were employed from ultrafast transient absorption spectroscopy to excited-state electronic structure calculations and quantum dynamical modelling. The result of our comprehensive study reveals the pivotal role of the LG as an EET booster through specific pathway guidance. In contrast, without the LG, the EET efficiency is reduced and the excitation energy predominantly dissipates within the BASHY chromophore. The present study highlights that LGs can actively contribute to optimizing the properties of dyad based systems and offers new design principles for monitoring uncaging via an intrinsic fluorescence readout.
Ionizable lipids such as the promising Dlin-MC3-DMA (MC3) are essential for the successful design of lipid nanoparticles (LNPs) as drug delivery agents. Combining molecular dynamics simulations with experimental data, such as neutron reflectivity experiments and other scattering techniques, is essential to provide insights into the internal structure of LNPs, which is not fully understood to date. However, the accuracy of the simulations relies on the choice of force field parameters and high-quality experimental data is indispensable to verify the parametrization. For MC3, different parameterizations in combination with the CHARMM and the Slipids force fields have recently emerged. Here, we complement the existing efforts by providing parameters for cationic and neutral MC3 compatible with the AMBER Lipid17 force field. Subsequently, we carefully assess the accuracy of the different force fields by providing a direct comparison to neutron reflectivity experiments of mixed lipid bilayers consisting of MC3 and DOPC at different pHs. At low pH (cationic MC3) and at high pH (neutral MC3) the newly developed MC3 parameters in combination with AMBER Lipid17 for DOPC give good agreement with the experiments. Overall, the agreement is similar compared to the Park-Im parameters for MC3 in combination with the CHARMM36 force field for DOPC. The Ermilova-Swenson MC3 parameters in combination with the Slipids force field underestimate the bilayer thickness. While the distribution of cationic MC3 is very similar, the different force fields for neutral MC3 reveal distinct differences ranging from strong accumulation in the membrane center (current MC3/AMBER Lipid17 DOPC), over mild accumulation (Park-Im MC3/CHARMM36 DOPC) to surface accumulation (Ermilova-Swenson MC3/Slipids DOPC). These pronounced differences highlight the importance of accurate force field parameters and their experimental validation.
We combine single-molecule Förster resonance energy transfer (single-molecule FRET) experiments with extensive all-atom molecular dynamics (MD) simulations (>100 μs) to characterize the conformational ensembles of single-stranded (ss) DNA and RNA in solution. From MD simulations with explicit dyes attached to single-stranded nucleic acids via flexible linkers, we calculate FRET efficiencies and fluorescence anisotropy decays. We find that dispersion-corrected water models alleviate the problem of overly abundant interactions between fluorescent dyes and the aromatic ring systems of nucleobases. To model dye motions in a computationally efficient and conformationally exhaustive manner, we introduce a dye-conformer library, built from simulations of dinucleotides with covalently attached dye molecules. We use this library to calculate FRET efficiencies for dT19, dA19, and rA19 simulated without explicit labels over a wide range of salt concentrations. For end-labeled homopolymeric pyrimidine ssDNA, MD simulations with the parmBSC1 force field capture the overall trend in salt-dependence of single-molecule FRET based distance measurements. For homopolymeric purine ssRNA and ssDNA, the DESRES and parmBSC1 force fields, respectively, provide useful starting points, even though our comparison also identifies clear deviations from experiment.
The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve the ultrafast structural changes in the carbonmonoxy myoglobin complex upon photolysis of the Fe-CO bond. Structural changes appear throughout the protein within 500 femtoseconds, with the C, F, and H helices moving away from the heme cofactor and the E and A helices moving toward it. These collective movements are predicted by hybrid quantum mechanics/molecular mechanics simulations. Together with the observed oscillations of residues contacting the heme, our calculations support the prediction that an immediate collective response of the protein occurs upon ligand dissociation, as a result of heme vibrational modes coupling to global modes of the protein.
Abstract We used EPR spectroscopy to characterize the structure of RNA duplexes and their internal twist, stretch and bending motions. We prepared eight 20‐base‐pair‐long RNA duplexes containing the rigid spin‐label Çm , a cytidine analogue, at two positions and acquired orientation‐selective PELDOR/DEER data. By using different frequency bands (X‐, Q‐, G‐band), detailed information about the distance and orientation of the labels was obtained and provided insights into the global conformational dynamics of the RNA duplex. We used 19 F Mims ENDOR experiments on three singly Çm ‐ and singly fluorine‐labeled RNA duplexes to determine the exact position of the Çm spin label in the helix. In a quantitative comparison to MD simulations of RNA with and without Çm spin labels, we found that state‐of‐the‐art force fields with explicit parameterization of the spin label were able to describe the conformational ensemble present in our experiments. The MD simulations further confirmed that the Çm spin labels are excellent mimics of cytidine inducing only small local changes in the RNA structure. Çm spin labels are thus ideally suited for high‐precision EPR experiments to probe the structure and, in conjunction with MD simulations, motions of RNA.
Abstract We used EPR spectroscopy to characterize the structure of RNA duplexes and their internal twist, stretch and bending motions. We prepared eight 20‐base‐pair‐long RNA duplexes containing the rigid spin‐label Çm , a cytidine analogue, at two positions and acquired orientation‐selective PELDOR/DEER data. By using different frequency bands (X‐, Q‐, G‐band), detailed information about the distance and orientation of the labels was obtained and provided insights into the global conformational dynamics of the RNA duplex. We used 19 F Mims ENDOR experiments on three singly Çm ‐ and singly fluorine‐labeled RNA duplexes to determine the exact position of the Çm spin label in the helix. In a quantitative comparison to MD simulations of RNA with and without Çm spin labels, we found that state‐of‐the‐art force fields with explicit parameterization of the spin label were able to describe the conformational ensemble present in our experiments. The MD simulations further confirmed that the Çm spin labels are excellent mimics of cytidine inducing only small local changes in the RNA structure. Çm spin labels are thus ideally suited for high‐precision EPR experiments to probe the structure and, in conjunction with MD simulations, motions of RNA.
Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.
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