Closing the Gap between Experiment and Simulation─A Holistic Study on the Complexation of Small Interfering RNAs with Polyethylenimine.

Rational design is pivotal in the modern development of nucleic acid nanocarrier systems. With the rising prominence of polymeric materials as alternatives to lipid-based carriers, understanding their structure-function relationships becomes paramount. Here, we introduce a newly developed coarse-grained model of polyethylenimine (PEI) based on the Martini 3 force field. This model facilitates molecular dynamics simulations of true-sized PEI molecules, exemplified by molecules with molecular weights of 1.3, 5, 10, and 25 kDa, with degrees of branching between 50.0 and 61.5%. We employed this model to investigate the thermodynamics of small interfering RNA (siRNA) complexation with PEI. Our simulations underscore the pivotal role of electrostatic interactions in the complexation process. Thermodynamic analyses revealed a stronger binding affinity with increased protonation, notably in acidic (endosomal) pH, compared to neutral conditions. Furthermore, the molecular weight of PEI was found to be a critical determinant of binding dynamics: smaller PEI molecules closely enveloped the siRNA, whereas larger ones extended outward, facilitating the formation of complexes with multiple RNA molecules. Experimental validations, encompassing isothermal titration calorimetry and single-molecule fluorescence spectroscopy, aligned well with our computational predictions. Our findings not only validate the fidelity of our PEI model but also accentuate the importance of in silico data in the rational design of polymeric drug carriers. The synergy between computational predictions and experimental validations, as showcased here, signals a refined and precise approach to drug carrier design.

DNA-Liposome Hybrid Carriers for Triggered Cargo Release.

The design of simple and versatile synthetic routes to accomplish triggered-release properties in carriers is of particular interest for drug delivery purposes. In this context, the programmability and adaptability of DNA nanoarchitectures in combination with liposomes have great potential to render biocompatible hybrid carriers for triggered cargo release. We present an approach to form a DNA mesh on large unilamellar liposomes incorporating a stimuli-responsive DNA building block. Upon incubation with a single-stranded DNA trigger sequence, a hairpin closes, and the DNA building block is allowed to self-contract. We demonstrate the actuation of this building block by single-molecule Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching, and fluorescence quenching measurements. By triggering this process, we demonstrate the elevated release of the dye calcein from the DNA-liposome hybrid carriers. Interestingly, the incubation of the doxorubicin-laden active hybrid carrier with HEK293T cells suggests increased cytotoxicity relative to a control carrier without the triggered-release mechanism. In the future, the trigger could be provided by peritumoral nucleic acid sequences and lead to site-selective release of encapsulated chemotherapeutics.

Single-Molecule Approved Surface Passivation.

Single-molecule experiments reveal structure function relationships and biomolecular dynamics in physiologically relevant conditions. In this issue of Structure, Park et al. (2020) report an optimized surface passivation strategy with polyethylene glycol in a dense, contracted conformation. Assembly of a functional transcription pre-initiation complex is demonstrated.

Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules.

We demonstrate measurements of the efficiency of competing Förster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.