Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e.

Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.

Long-Timescale Simulations Revealed Critical Non-Conserved Residues of Phosphodiesterases Affecting Selectivity of BAY60-7550.

A major obstacle of the selective inhibitor design for specific human phosphodiesterase (PDE) is that highly conserved catalytic pockets are difficult to be distinguished by inhibitor molecules. To overcome this, a feasible path is to understand the molecular determinants underlying the selectivity of current inhibitors. BAY60-7550 (BAY for short; IC50 = 4.7 nM) is a highly selective inhibitor targeting PDE2A which is a dual-specificity PDE and an attractive target for therapeutic intervention of the central nervous system (CNS) disorders. Recent studies suggest that molecular determinants may be in binding processes of BAY. However, a detailed understanding of these processes are still lacking. To explore these processes, High-Throughput Molecular Dynamics (HTMD) simulations were performed to reproduce the spontaneous association of BAY with catalytic pockets of 4 PDE isoforms; Ligand Gaussian Accelerated Molecular Dynamics (LiGaMD) simulations were performed to reproduce the unbinding-rebinding processes of FKG and MC2, two pyrazolopyrimidinone PDE2A selective inhibitors, in the PDE2A system. The produced molecular trajectories were analyzed by the Markov state model (MSM) and the molecular mechanics/generalized Born surface area (MM/GBSA). The results showed that the non-covalent interactions between the non-conserved residues and BAY, especially the hydrogen bonds, determined the unique binding pathways of BAY on the surface of PDE2A. These pathways were different from those of BAY on the surface of the other three PDE isoforms and the binding pathways of the other two PDE2A inhibitors in PDE2A systems. These differences were ultimately reflected in the high selectivity of this inhibitor for PDE2A. As a result, this study demonstrates the critical role of the binding processes in the selectivity of BAY, and also identifies the key non-conserved residues affecting the binding processes of BAY. Thus, this study provides a new perspective and data support for the further development of BAY-derived inhibitors targeting PDE2A.

Optimization of Gonyautoxin1/4-Binding G-Quadruplex Aptamers by Label-Free Surface-Enhanced Raman Spectroscopy.

Nucleic acids with G-quadruplex (G4) structures play an important role in physiological function, analysis and detection, clinical diagnosis and treatment, and new drug research and development. Aptamers obtained using systematic evolution of ligands via exponential enrichment (SELEX) screening technology do not always have the best affinity or binding specificity to ligands. Therefore, the establishment of a structure-oriented experimental method is of great significance. To study the potential of surface-enhanced Raman spectroscopy (SERS) in aptamer optimization, marine biotoxin gonyautoxin (GTX)1/4 and its G4 aptamer obtained using SELEX were selected. The binding site and the induced fit of the aptamer to GTX1/4 were confirmed using SERS combined with two-dimensional correlation spectroscopy. The intensity of interaction between GTX1/4 and G4 was also quantified by measuring the relative intensity of SERS bands corresponding to intramolecular hydrogen bonds. Furthermore, the interaction between GTX1/4 and optimized aptamers was analyzed. The order of intensity change in the characteristic bands of G4 aptamers was consistent with the order of affinity calculated using microscale thermophoresis and molecular dynamics simulations. SERS provides a rapid, sensitive, and economical post-SELEX optimization of aptamers. It is also a reference for future research on other nucleic acid sequences containing G4 structures.

De novo design of DNA aptamers that target okadaic acid (OA) by docking-then-assembling of single nucleotides.

Okadaic acid (OA) is a diarrhetic shellfish poison widespread in ocean, so its detection is of great significance to seafood safety. Because of good sensitivity and low cost, biosensors using nucleic-acid aptamers as the recognition molecules are emerging as an important detection tool. However, the traditional SELEX screening method for acquiring OA high-affinity aptamers is time- and resource-intensive. Alternatively, here we developed a de novo design method based on the 3D structure of a target molecule, such as OA. Without experimental screening, this method designs OA aptamers by a computational approach of docking-then-assembling (DTA) of single nucleotides (A, C, G and T) as: (1) determining the high-affinity nucleotide binding sites of the target molecule via saturated molecular docking; (2) assembling the bound nucleotides into binding units to the target molecule; (3) constructing full-length aptamers by introducing stabilizing units to connect these binding units. In this way, five OA aptamers were designed, and microscale thermophoresis (MST) experiments verified that their Kd values are in the range of 100-600 nM; and one of them (named 9CGAT_4_a) could specifically bind to OA with low affinities for the other three marine biotoxins. Therefore, this study provides high-affinity and specific aptamers for the development of OA biosensors, and presents a promising de novo design method applicable to other target molecules.

Repurposing of thermally stable nucleic-acid aptamers for targeting tetrodotoxin (TTX).

Tetrodotoxin (TTX) is a lethal neurotoxin produced by the endosymbiotic bacteria in the gut of puffer fish. Currently, there is no effective and economical method to detect TTX, so it is very interesting to develop low-cost and high-sensitivity detection methods by using nucleic-acid aptamers as the recognition molecules. However, traditional SELEX screening of aptamers for targeting small molecules such as TTX is labor-intensive, and usually the success rate is low. Here, we employed a strategy of "repurposing old aptamers for new uses" to develop high-affinity aptamers for TTX. To this end, we first collected thermally stable DNA aptamers and predicted their affinities for TTX by molecular docking; then, we identified high-affinity candidates and verified them by microscale thermophoresis (MST) experiments. In this way, two thermally stable aptamers (Tv-51 and AI-57) were found to possess nanomolar affinities for TTX. Moreover, we performed spontaneous binding simulations to reveal their binding mechanisms to TTX and thereby identified the key bases for the binding. Guided by these, two variants (Tv-46 and AI-52) with higher affinities and specificities were subsequently engineered and confirmed by the MST experiments. So, this study not only provides potential recognition molecules for the technology developments of TTX detection, but also demonstrates an effective repurposing approach to the discovery of high-affinity aptamers for new target molecules.

Spontaneous binding of potential COVID-19 drugs (Camostat and Nafamostat) to human serine protease TMPRSS2.

Effective treatment or vaccine is not yet available for combating SARS coronavirus 2 (SARS-CoV-2) that caused the COVID-19 pandemic. Recent studies showed that two drugs, Camostat and Nafamostat, might be repurposed to treat COVID-19 by inhibiting human TMPRSS2 required for proteolytic activation of viral spike (S) glycoprotein. However, their molecular mechanisms of pharmacological action remain unclear. Here, we perform molecular dynamics simulations to investigate their native binding sites on TMPRSS2. We revealed that both drugs could spontaneously and stably bind to the TMPRSS2 catalytic center, and thereby inhibit its proteolytic processing of the S protein. Also, we found that Nafamostat is more specific than Camostat for binding to the catalytic center, consistent with reported observation that Nafamostat blocks the SARS-CoV-2 infection at a lower concentration. Thus, this study provides mechanistic insights into the Camostat and Nafamostat inhibition of the SARS-CoV-2 infection, and offers useful information for COVID-19 drug development.

De novo post-SELEX optimization of a G-quadruplex DNA aptamer binding to marine toxin gonyautoxin 1/4.

Ligand-binding aptamers obtained by SELEX (Systematic Evolution of Ligands by EXponential enrichment) often have low affinity or/and specificity, and post-SELEX optimization is usually needed. Due to experimental difficulty in determining three-dimensional (3D) structures of aptamer-ligand complexes, there are few structure-guided methods for rational post-SELEX optimization. Here, we employed a de novo optimization approach to engineer high-affinity variants for a G-quadruplex (GQ) aptamer (GO18-T-d) that specifically binds to marine toxin gonyautoxin 1/4 (GTX1/4). First, temperature-dependent modeling was carried out to predict the atomic structure of GO18-T-d. Then, to identify key bases for the optimization, spontaneous binding simulations were performed to reveal the complex structure of GO18-T-d with GTX1/4. Finally, binding energy analysis was conducted to evaluate the designed variants for high affinity. We predicted that GO18-T-d has the typical parallel GQ topology, consistent with circular dichroism (CD) measurements. Our simulations showed that the 5'-end of GO18-T-d hinders the GTX1/4 movement toward the binding pocket, leading to a designed variant that removes the first 5 nucleotides at the 5'-end. Microscale thermophoresis (MST) experiments verified that the binding affinity of the engineered aptamer increases by ~20 folds. Thus, this study not only provides a high-affinity variant of GO18-T-d, but also suggests that our computational approach is useful for the structure-guided optimization of GQ aptamers.

Identifying conformational changes of aptamer binding to theophylline: A combined biolayer interferometry, surface-enhanced Raman spectroscopy, and molecular dynamics study.

Theophylline is a potent bronchodilator for the treatment of asthma, bronchitis, and emphysema. Its narrow therapeutic window (20-100 µM) demands that the blood concentration of theophylline be monitored carefully, which can be achieved by aptamer capture. Thus, an understanding of what occurs when aptamers bind to theophylline is critical for identifying a high-affinity and high-specificity aptamer, which improve the sensitivity and selectivity of theophylline detection. Consequently, there is an urgent need to develop a simple, convenient, and nondestructive method to monitor conformational changes during the binding process. Here, we report the determination of the affinity of a selected aptamer and theophylline via biolayer interferometry (BLI) experiments. Additionally, using surface-enhanced Raman spectroscopy (SERS), the conformational changes on theophylline-aptamer binding were identified from differences in the SER spectra. Finally, molecular dynamics (MD) simulations were used to identify the specific conformational changes of the aptamer during the binding process. Such a combined BLI-SERS-MD method provides an in-depth understanding of the theophylline-aptamer binding processes and a comprehensive explanation for conformational changes, which helps to select, design, and modify an aptamer with high affinity and specificity. It can also be used as a scheme for the study of other aptamer-ligand interactions, which can be applied to the detection, sensing, clinical diagnosis, and treatment of diseases.

Exploring the Mechanism of Inhibition of Au Nanoparticles on the Aggregation of Amyloid-ß(16-22) Peptides at the Atom Level by All-Atom Molecular Dynamics.

The abnormal self-assembly of the amyloid-ß peptide into toxic ß-rich aggregates can cause Alzheimer's disease. Recently, it has been shown that small gold nanoparticles (AuNPs) inhibit Aß aggregation and fibrillation by slowing down the nucleation process in experimental studies. However, the effects of AuNPs on Aß oligomeric structures are still unclear. In this study, we investigate the conformation of Aß(16-22) tetramers/octamers in the absence and presence of AuNPs using extensive all-atom molecular-dynamics simulations in explicit solvent. Our studies demonstrate that the addition of AuNPs into Aß(16-22) solution prevents ß-sheet formation, and the inhibition depends on the concentration of Aß(16-22) peptides. A detailed analysis of the Aß(16-22)/Aß(16-22)/water/AuNPs interactions reveals that AuNPs inhibit the ß-sheet formation resulting from the same physical forces: hydrophobic interactions. Overall, our computational study provides evidence that AuNPs are likely to inhibit Aß(16-22) and full-length Aß fibrillation. Thus, this work provides theoretical insights into the development of inorganic nanoparticles as drug candidates for treatment of AD.

Three-dimensional reconstruction of anomalous eutectic in laser remelted Ni-30 wt.% Sn alloy.

Laser remelting has been performed on Ni-30 wt.% Sn hypoeutectic alloy. An anomalous eutectic formed at the bottom of the molten pool when the sample was remelted thoroughly. 3D morphologies of the α-Ni and Ni3Sn phases in the anomalous eutectic region were obtained and investigated using serial sectioning reconstruction technology. It is found that the Ni3Sn phase has a continuous interconnected network structure and the α-Ni phase is distributed as separate particles in the anomalous eutectic, which is consistent with the electron backscatter diffraction pattern examinations. The α-Ni particles in the anomalous eutectic are supersaturated with Sn element as compared with the equilibrium phase diagram. Meanwhile, small wavy lamella eutectics coexist with anomalous eutectics. The Trivedi-Magnin-Kurz model was used to estimate undercooling with lamellar spacing. The results suggest that the critical undercooling found in undercooling solidification is not a sufficient condition for anomalous eutectic formation. Besides, α-Ni particles in the anomalous eutectic do not exhibit a completely random misorientation and some neighboring α-Ni particles have the same orientation. It is shown that both the coupled and decoupled growth of the eutectic two phases can generate the α-Ni + Ni3Sn anomalous eutectic structure.