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.

Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy.

3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques with fluorescence imaging. Here, we achieve 3D super-resolution by combining the 2D localization of pMINFLUX with the axial information of graphene energy transfer (GET) and the single-molecule switching by DNA-PAINT. We demonstrate <2 nm localization precision in all 3 dimension with axial precision reaching below 0.3 nm. In 3D DNA-PAINT measurements, structural features, i.e., individual docking strands at distances of 3 nm, are directly resolved on DNA origami structures. pMINFLUX and GET represent a particular synergetic combination for super-resolution imaging near the surface such as for cell adhesion and membrane complexes as the information of each photon is used for both 2D and axial localization information. Furthermore, we introduce local PAINT (L-PAINT), in which DNA-PAINT imager strands are equipped with an additional binding sequence for local upconcentration improving signal-to-background ratio and imaging speed of local clusters. L-PAINT is demonstrated by imaging a triangular structure with 6 nm side lengths within seconds.

Graphene Energy Transfer for Single-Molecule Biophysics, Biosensing, and Super-Resolution Microscopy.

Graphene is considered a game-changing material, especially for its mechanical and electrical properties. This work exploits that graphene is almost transparent but quenches fluorescence in a range up to ≈40 nm. Graphene as a broadband and unbleachable energy-transfer acceptor without labeling, is used to precisely determine the height of molecules with respect to graphene, to visualize the dynamics of DNA nanostructures, and to determine the orientation of Förster-type resonance energy transfer (FRET) pairs. Using DNA origami nanopositioners, biosensing, single-molecule tracking, and DNA PAINT super-resolution with <3 nm z-resolution are demonstrated. The range of examples shows the potential of graphene-on-glass coverslips as a versatile platform for single-molecule biophysics, biosensing, and super-resolution microscopy.

Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope.

The advent of highly sensitive photodetectors and the development of photostabilization strategies made detecting the fluorescence of single molecules a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g., in point-of-care diagnostic settings, where costly equipment would be prohibitive. Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in NACHOS emit up to 461-fold (average of 89 ± 7-fold) brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia on the road.

Pulsed Interleaved MINFLUX.

We introduce p-MINFLUX, a new implementation of the highly photon-efficient single-molecule localization method with a simplified experimental setup and additional fluorescence lifetime information. In contrast to the original MINFLUX implementation, p-MINFLUX uses interleaved laser pulses to deliver the doughnut-shaped excitation foci at a maximum repetition rate. Using both static and dynamic DNA origami model systems, we demonstrate the performance of p-MINFLUX for single-molecule localization nanoscopy and tracking, respectively. p-MINFLUX delivers 1-2 nm localization precision with 2000-1000 photon counts. In addition, p-MINFLUX gives access to the fluorescence lifetime enabling multiplexing and super-resolved lifetime imaging. p-MINFLUX should help to unlock the full potential of innovative single-molecule localization schemes.