Probing Intermolecular Interactions within the Amyloid β Trimer Using a Tethered Polymer Nanoarray

Amyloid oligomers are considered the most neurotoxic species of amyloid aggregates. Spontaneous assembly of amyloids into aggregates is recognized as a major molecular mechanism behind Alzheimer’s disease and other neurodegenerative disorders involving protein aggregation. Characterization of such oligomers is extremely challenging but complicated by their transient nature. Previously, we introduced a flexible nanoarray (FNA) method enabling us to probe dimers assembled by the amyloid β (14-23) [Aβ (14-23)] peptide. The study presented herein modifies and enhances this approach to assemble and probe trimers of Aβ (14-23). A metal-free click chemistry approach was used, in which dibenzocyclooctyne (DBCO) groups were incorporated at selected sites within the FNA template to click Aβ (14-23) monomers at their terminal azide groups. Atomic force microscopy (AFM) force spectroscopy was employed to characterize the assemblies. The force measurement data demonstrate that the dissociation of the trimer undergoes a stepwise pattern, in which the first monomer dissociates at the rupture force ∼48 ± 2.4 pN. The remaining dimer ruptures at the second step at a slightly larger rupture force (∼53 ± 3.2 pN). The assembled trimer was found to be quite dynamic, and transient species of this inherently dynamic process were identified

Molecular Mechanism of Misfolding and Aggregation of Aβ(13–23)

The misfolding and self-assembly of the amyloid-beta (Aβ) peptide into aggregates is a molecular signature of the development of Alzheimer’s disease, but molecular mechanisms of the peptide aggregation remain unknown. Here, we combined Atomic Force Microscopy (AFM) and Molecular Dynamics (MD) simulations to characterize the misfolding process of an Aβ peptide. Dynamic force spectroscopy AFM analysis showed that the peptide forms stable dimers with a lifetime of ∼1 s. During MD simulations, isolated monomers gradually adopt essentially similar nonstructured conformations independent from the initial structure. However, when two monomers approach their structure changes dramatically, and the conformational space for the two monomers become restricted. The arrangement of monomers in antiparallel orientation leads to the cooperative formation of β-sheet conformation. Interactions, including hydrogen bonds, salt bridges, and weakly polar interactions of side chains stabilize the structure of the dimer. Under the applied force, the dimer, as during the AFM experiments, dissociates in a cooperative manner. Thus, misfolding of the Aβ peptide proceeds via the loss of conformational flexibility and formation of stable dimers suggesting their key role in the subsequent Aβ aggregation process

Physicochemically Tunable Polyfunctionalized RNA Square Architecture with Fluorogenic and Ribozymatic Properties

Recent advances in RNA nanotechnology allow the rational design of various nanoarchitectures. Previous methods utilized conserved angles from natural RNA motifs to form geometries with specific sizes. However, the feasibility of producing RNA architecture with variable sizes using native motifs featuring fixed sizes and angles is limited. It would be advantageous to display RNA nanoparticles of diverse shape and size derived from a given primary sequence. Here, we report an approach to construct RNA nanoparticles with tunable size and stability. Multifunctional RNA squares with a 90° angle were constructed by tuning the 60° angle of the three-way junction (3WJ) motif from the packaging RNA (pRNA) of the bacteriophage phi29 DNA packaging motor. The physicochemical properties and size of the RNA square were also easily tuned by modulating the “core” strand and adjusting the length of the sides of the square <i>via</i> predictable design. Squares of 5, 10, and 20 nm were constructed, each showing diverse thermodynamic and chemical stabilities. Four “arms” extending from the corners of the square were used to incorporate siRNA, ribozyme, and fluorogenic RNA motifs. Unique intramolecular contact using the pre-existing intricacy of the 3WJ avoids relatively weaker intermolecular interactions <i>via</i> kissing loops or sticky ends. Utilizing the 3WJ motif, we have employed a modular design technique to construct variable-size RNA squares with controllable properties and functionalities for diverse and versatile applications with engineering, pharmaceutical, and medical potential. This technique for simple design to finely tune physicochemical properties adds a new angle to RNA nanotechnology

Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that <i>Escherichia coli</i> SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg²⁺ cations or a high ionic strength. In the absence of Mg²⁺ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg²⁺ cations

Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that <i>Escherichia coli</i> SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg²⁺ cations or a high ionic strength. In the absence of Mg²⁺ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg²⁺ cations

Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that <i>Escherichia coli</i> SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg²⁺ cations or a high ionic strength. In the absence of Mg²⁺ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg²⁺ cations

CIFOR Poverty and Environment Network (PEN)

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of <i>Escherichia coli</i> RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks

Dynamics of the Interaction of RecG Protein with Stalled Replication Forks

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of <i>Escherichia coli</i> RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks

Dynamics of the Interaction of RecG Protein with Stalled Replication Forks

As a guardian of the bacterial genome, the RecG DNA helicase repairs DNA replication and rescues stalled replication. We applied atomic force microscopy (AFM) to directly visualize dynamics of RecG upon the interaction with replication fork substrates in the presence and absence of SSB using high-speed AFM. We directly visualized that RecG moves back and forth over dozens of base pairs in the presence of SSB. There is no RecG translocation in the absence of SSB. Computational modeling was performed to build models of <i>Escherichia coli</i> RecG in a free state and in complex with the fork. The simulations revealed the formation of complexes of RecG with the fork and identified conformational transitions that may be responsible for RecG remodeling that can facilitate RecG translocation along the DNA duplex. Such complexes do not form with the DNA duplex, which is in line with experimental data. Overall, our results provide mechanistic insights into the modes of interaction of RecG with the replication fork, suggesting a novel role of RecG in the repair of stalled DNA replication forks

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

29 janvier 19431943/01/29 (A44,N15335)