Oligomer Dynamics of LL-37 Truncated Fragments Probed by α-Hemolysin Pore and Molecular Simulations.

Oligomerization of antimicrobial peptides (AMPs) is critical in their effects on pathogens. LL-37 and its truncated fragments are widely investigated regarding their structures, antimicrobial activities, and application, such as developing new antibiotics. Due to the small size and weak intermolecular interactions of LL-37 fragments, it is still elusive to establish the relationship between oligomeric states and antimicrobial activities. Here, an α-hemolysin nanopore, mass spectrometry (MS), and molecular dynamic (MD) simulations are used to characterize the oligomeric states of two LL-37 fragments. Nanopore studies provide evidence of trapping events related to the oligomer formation and provide further details on their stabilities, which are confirmed by MS and MD simulations. Furthermore, simulation results reveal the molecular basis of oligomer dynamics and states of LL-37 fragments. This work provides unique insights into the relationship between the oligomer dynamics of AMPs and their antimicrobial activities at the single-molecule level. The study demonstrates how integrating methods allows deciphering single molecule level understanding from nanopore sensing approaches.

Visualizing RNA Structures by SAXS-Driven MD Simulations.

The biological role of biomolecules is intimately linked to their structural dynamics. Experimental or computational techniques alone are often insufficient to determine accurate structural ensembles in atomic detail. We use all-atom molecular dynamics (MD) simulations and couple it to small-angle X-ray scattering (SAXS) experiments to resolve the structural dynamics of RNA molecules. To accomplish this task, we utilize a set of re-weighting and biasing techniques tailored for RNA molecules. To showcase our approach, we study two RNA molecules: a riboswitch that shows structural variations upon ligand binding, and a two-way junction RNA that displays structural heterogeneity and sensitivity to salt conditions. Integration of MD simulations and experiments allows the accurate construction of conformational ensembles of RNA molecules. We observe a dynamic change of the SAM-I riboswitch conformations depending on its binding partners. The binding of SAM and Mg2+ cations stabilizes the compact state. The absence of Mg2+ or SAM leads to the loss of tertiary contacts, resulting in a dramatic expansion of the riboswitch conformations. The sensitivity of RNA structures to the ionic strength demonstrates itself in the helix junction helix (HJH). The HJH shows non-monotonic compaction as the ionic strength increases. The physics-based picture derived from the experimentally guided MD simulations allows biophysical characterization of RNA molecules. All in all, SAXS-guided MD simulations offer great prospects for studying RNA structural dynamics.

Differences in ion-RNA binding modes due to charge density variations explain the stability of RNA in monovalent salts.

The stability of RNA increases as the charge density of the alkali metal cations increases. The molecular mechanism for this phenomenon remains elusive. To fill this gap, we performed all-atom molecular dynamics pulling simulations of HIV-1 trans-activation response RNA. We first established that the free energy landscape obtained in the simulations is in excellent agreement with the single-molecule optical tweezer experiments. The origin of the stronger stability in sodium compared to potassium is found to be due to the differences in the charge density-related binding modes. The smaller hydrated sodium ion preferentially binds to the highly charged phosphates that have high surface area. In contrast, the larger potassium ions interact with the major grooves. As a result, more cations condense around phosphate groups in the case of sodium ions, leading to the reduction of electrostatic repulsion. Because the proposed mechanism is generic, we predict that the same conclusions are valid for divalent alkaline earth metal cations.

Peptidomimetic-Based Multidomain Targeting Offers Critical Evaluation of Aß Structure and Toxic Function.

The prevailing hypothesis stipulates that the preamyloid oligomers of Aß are the main culprits associated with the onset and progression of Alzheimer's disease (AD), which has prompted efforts to search for therapeutic agents with the ability to inhibit Aß oligomerization and amyloidogenesis. However, clinical progress is impeded by the limited structural information about the neurotoxic oligomers. To address this issue, we have adopted a synthetic approach, where a library of oligopyridylamide-based small molecules was tested against various microscopic events implicated in the self-assembly of Aß. Two oligopyridylamides bind to different domains of Aß and affect distinct microscopic events in Aß self-assembly. The study lays the foundations for a dual recognition strategy to simultaneously target different domains of Aß for further improvement in antiamyloidogenic activity. The data demonstrate that one of the most effective oligopyridylamides forms a high affinity complex with Aß, which sustains the compound's activity in cellular milieu. The oligopyridylamide was able to rescue cells when introduced 24 h after the incubation of Aß. The rescue of Aß toxicity is potentially a consequence of the colocalization of the oligopyridylamide with Aß. The synthetic tools utilized here provide a straightforward strategic framework to identify a range of potent antagonists of Aß-mediated toxic functions. This approach could be a powerful route to the design of candidate drugs for various amyloid diseases that have so far proven to be "untargetable".

DNA-assisted oligomerization of pore-forming toxin monomers into precisely-controlled protein channels.

We have developed a novel approach for creating membrane-spanning protein-based pores. The construction principle is based on using well-defined, circular DNA nanostructures to arrange a precise number of pore-forming protein toxin monomers. We can thereby obtain, for the first time, protein pores with specifically set diameters. We demonstrate this principle by constructing artificial alpha-hemolysin (αHL) pores. The DNA/αHL hybrid nanopores composed of twelve, twenty or twenty-six monomers show stable insertions into lipid bilayers during electrical recordings, along with steady, pore size-dependent current levels. Our approach successfully advances the applicability of nanopores, in particular towards label-free studies of single molecules in large nanoscaled biological structures.

Foldamer-Mediated Structural Rearrangement Attenuates Aß Oligomerization and Cytotoxicity.

The conversion of the native random coil amyloid beta (Aß) into amyloid fibers is thought to be a key event in the progression of Alzheimer's disease (AD). A significant body of evidence suggests that the highly dynamic Aß oligomers are the main causal agent associated with the onset of AD. Among many potential therapeutic approaches, one is the modulation of Aß conformation into off-pathway structures to avoid the formation of the putative neurotoxic Aß oligomers. A library of oligoquinolines was screened to identify antagonists of Aß oligomerization, amyloid formation, and cytotoxicity. A dianionic tetraquinoline, denoted as 5, was one of the most potent antagonists of Aß fibrillation. Biophysical assays including amyloid kinetics, dot blot, ELISA, and TEM show that 5 effectively inhibits both Aß oligomerization and fibrillation. The antagonist activity of 5 toward Aß aggregation diminishes with sequence and positional changes in the surface functionalities. 5 binds to the central discordant α-helical region and induces a unique α-helical conformation in Aß. Interestingly, 5 adjusts its conformation to optimize the antagonist activity against Aß. 5 effectively rescues neuroblastoma cells from Aß-mediated cytotoxicity and antagonizes fibrillation and cytotoxicity pathways of secondary nucleation induced by seeding. 5 is also equally effective in inhibiting preformed oligomer-mediated processes. Collectively, 5 induces strong secondary structure in Aß and inhibits its functions including oligomerization, fibrillation, and cytotoxicity.

Dielectrophoresis of gold nanoparticles conjugated to DNA origami structures.

DNA nanostructures are promising construction materials to bridge the gap between self-assembly of functional molecules and conventional top-down fabrication methods in nanotechnology. Their positioning onto specific locations of a microstructured substrate is an important task towards this aim. Here we study manipulation and positioning of pristine and of gold nanoparticle-conjugated tubular DNA origami structures using ac dielectrophoresis. The dielectrophoretic behavior was investigated employing fluorescence microscopy. For the pristine origami, a significant dielectrophoretic response was found to take place in the megahertz range, whereas, due to the higher polarizability of the metallic nanoparticles, the nanoparticle/DNA hybrid structures required a lower electrical field strength and frequency for a comparable trapping at the edges of the electrode structure. The nanoparticle conjugation additionally resulted in a remarkable alteration of the DNA structure arrangement. The growth of linear, chain-like structures in between electrodes at applied frequencies in the megahertz range was observed. The long-range chain formation is caused by a local, gold nanoparticle-induced field concentration along the DNA nanostructures, which in turn, creates dielectrophoretic forces that enable the observed self-alignment of the hybrid structures.

Methods to Characterize the Oligonucleotide Functionalization of Quantum Dots.

Currently, DNA nanotechnology offers the most programmable, scalable, and accurate route for the self-assembly of matter with nanometer precision into 1, 2, or 3D structures. One example is DNA origami that is well suited to serve as a molecularly defined "breadboard", and thus, to organize various nanomaterials such as nanoparticles into hybrid systems. Since the controlled assembly of quantum dots (QDs) is of high interest in the field of photonics and other optoelectronic applications, a more detailed view on the functionalization of QDs with oligonucleotides shall be achieved. In this work, four different methods are presented to characterize the functionalization of thiol-capped cadmium telluride QDs with oligonucleotides and for the precise quantification of the number of oligonucleotides bound to the QD surface. This study enables applications requiring the self-assembly of semiconductor-oligonucleotide hybrid materials and proves the conjugation success in a simple and straightforward manner.