Preferred Binding Sites Research Articles

Characterization of Copper(II) and Zinc(II) Complexes of Peptides Mimicking the CuZnSOD Enzyme.

Antimicrobial peptides are short cationic peptides that are present on biological surfaces susceptible to infection, and they play an important role in innate immunity. These peptides, like other compounds with antimicrobial activity, often have significant superoxide dismutase (SOD) activity. One direction of our research is the characterization of peptides modeling the CuZnSOD enzyme and the determination of their biological activity, and these results may contribute to the development of novel antimicrobial peptides. In the framework of this research, we have synthesized 10, 15, and 16-membered model peptides containing the amino acid sequence corresponding to the Cu(II) and Zn(II) binding sites of the CuZnSOD enzyme, namely the Zn(II)-binding HVGD sequence (80-83. fragments), the Cu(II)-binding sequence HVH (fragments 46-48), and the histidine (His63), which links the two metal ions as an imidazolate bridge: Ac-FHVHEGPHFN-NH2 (L1(10)), Ac-FHVHAGPHFNGGHVG-NH2 (L2(15)), and Ac-FHVHEGPHFNGGHVGD-NH2 (L3(16)). pH-potentiometric, UV-Vis-, and CD-spectroscopy studies of the Cu(II), Zn(II), and Cu(II)-Zn(II) mixed complexes of these peptides were performed, and the SOD activity of the complexes was determined. The binding sites preferred by Cu(II) and Zn(II) were identified by means of CD-spectroscopy. From the results obtained for these systems, it can be concluded that in equimolar solution, the -(NGG)HVGD- sequence of the peptides is the preferred binding site for copper(II) ion. However, in the presence of both metal ions, according to the native enzyme, the -HVGD- sequence offers the main binding site for Zn(II), while the majority of Cu(II) binds to the -FHVH- sequence. Based on the SOD activity assays, complexes of the 15- and 16-membered peptide have a significant SOD activity. Although this activity is smaller than that of the native CuZnSOD enzyme, the complexes showed better performance in the degradation of superoxide anion than other SOD mimics. Thus, the incorporation of specific amino acid sequences mimicking the CuZnSOD enzyme increases the efficiency of model systems in the catalytic decomposition of superoxide anion.

Unveiling the molecular interaction of hepatitis B virus inhibitor, entecavir with human serum albumin through computational, microscopic and spectroscopic approaches

Molecular docking, molecular dynamics (MD) simulation, atomic force microscopy (AFM) and multi-spectroscopic techniques were selected to unveil the molecular association between the hepatitis B virus (HBV) inhibitor, entecavir (ETR), and the major blood plasma transporter, human serum albumin (HSA). The entire docking and simulation analyses recognized ETR binding to subdomain IIA (Site I) of HSA through hydrogen bonds, hydrophobic and van der Waals forces while maintaining the complex’s stability throughout the 100 ns. A gradual lessening in the Stern-Volmer quenching constant ( K sv ) with rising temperatures registered ETR-induced quenching of HBV fluorescence as static quenching, thus advising complexation between ETR and HSA. The further advocation of this conclusion was seen from a larger value of the biomolecular quenching rate constant (( kq ) > 1010 M−1s−1), changes in the spectra (UV-Vis absorption) of HSA following ETR inclusion and ETR-induced swelling of HSA in the AFM results. The ETR appeared to bind to HSA with moderate affinity ( K a = 1.87 − 1.19 × 10 4 M−1) at 290, 300 and 310 K. Significant alterations in the protein’s secondary and tertiary structures, including changes in the protein’s Tyr/Trp microenvironment, were also detected by circular dichroism and three-dimensional fluorescence spectra when the protein was bound to ETR. The findings of the drug displacement study backed the docking results of Site I as ETR's preferred binding site in HSA. Communicated by Ramaswamy H. Sarma

On assessing the carbon capture performance of graphynes with particle swarm optimization.

Tackling climate change is one of the greatest challenges of current times and therefore the development of efficient technologies to limit anthropogenic emissions is of utmost urgency. Recent research towards this goal has alluded to the use of carbon-based solid sorbents for carbon capture. Graphynes (GYs), an interesting class of porous carbon membranes, have recently proven their potential as excellent membranes for gas adsorption and separation. Herein, we explored the CO2 and N2 adsorption characteristics and CO2/N2 selectivities of a class of GYs, namely γ-GY-1, γ-GY-2 and γ-GY-4. We investigated the putative global minimum geometries of adsorbed unary (n = 2-10) and binary (n : m; n, m ∈ [1, 8]) clusters of CO2 and N2 by employing a stochastic global optimization method called particle swarm optimization in conjunction with empirical intermolecular force field formulations. The intervening interactions are modeled using various pairwise potentials, including Lennard-Jones potential, improved Lennard-Jones potential, Buckingham potential and Coulombic potential. The binding energies for both unary and binary clusters are highest for adsorption on γ-GY-1, followed by γ-GY-2. The putative global minimum geometries suggested that N2 molecules preferred binding over the pore centres while CO2 molecules showed higher clustering propensity than any binding site preference. The predicted interaction energies suggested higher selectivity for CO2 over N2 for all the three γ-GYs.

Probing the pH-dependency of DC-SIGN/R multivalent lectin-glycan interactions using polyvalent glycan-gold nanoparticles.

The dendritic cell tetrameric lectin, DC-SIGN, and its closely related endothelial cell lectin, DC-SIGNR (collectively abbreviated as DC-SIGN/R) play a key role in the binding and transmission of deadly viruses, including Ebola, HIV, HCV, and SARS-CoV-2. Their virus binding/release processes involve a gradually acidifying environment following the natural intracellular trafficking pathways. Therefore, understanding DC-SIGN/R's pH-dependent binding properties with glycan ligands is of great importance. We have recently developed densely glycosylated gold nanoparticles (glycan-GNPs) as a powerful new tool for probing DC-SIGN/R multivalent lectin-glycan interaction (MLGI) mechanisms. They can provide not only quantitative MLGI affinities but also important structural information, such as binding site orientation and binding modes. Herein, we further employ the glycan-GNP probes to investigate the pH dependency of DC-SIGN/R MLGI properties. We find that DC-SIGN/R MLGIs exhibit distinct pH dependence over the normal physiological (7.4) to lysosomal (∼4.6) pH range. DC-SIGN binds glycan-GNPs strongly and stably from pH 7.4 to ∼5.8, but the binding is weakened significantly as pH decreases to ≤5.4 and may be fully dissociated at pH 4.6. This behaviour is fully consistent with DC-SIGN's role as an endocytic recycling receptor. In contrast, DC-SIGNR's affinity with glycan-GNPs is enhanced with the decreasing pH from 7.4 to 5.4, peaking at pH 5.4, and then reduced as pH is further lowered. Interestingly, both DC-SIGN/R binding with glycan-GNPs are found to be partially reversible in a pH-dependent manner.

Probing scaffold size effects on multivalent lectin-glycan binding affinity, thermodynamics and antiviral properties using polyvalent glycan-gold nanoparticles.

Multivalent lectin-glycan interactions (MLGIs) are pivotal for viral infections and immune regulation. Their structural and biophysical data are thus highly valuable, not only for understanding their basic mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information for some important MGLIs remains poorly understood, greatly limiting research progress. We have recently developed densely glycosylated nanoparticles, e.g., ∼4 nm quantum dots (QDs) or ∼5 nm gold nanoparticles (GNPs), as mechanistic probes for MLGIs. Using two important model lectin viral receptors, DC-SIGN and DC-SIGNR, we have shown that these probes can not only offer sensitive fluorescence assays for quantifying MLGI affinities, but also reveal key structural information (e.g., binding site orientation and binding mode) useful for MLGI targeting. However, the small sizes of the previous scaffolds may not be optimal for maximising MLGI affinity and targeting specificity. Herein, using α-manno-α-1,2-biose (DiMan) functionalised GNP (GNP-DiMan) probes, we have systematically studied how GNP scaffold size (e.g., 5, 13, and 27 nm) and glycan density (e.g., 100, 75, 50 and 25%) determine their MLGI affinities, thermodynamics, and antiviral properties. We have developed a new GNP fluorescence quenching assay format to minimise the possible interference of GNP's strong inner filter effect in MLGI affinity quantification, revealing that increasing the GNP size is highly beneficial for enhancing MLGI affinity. We have further determined the MLGI thermodynamics by combining temperature-dependent affinity and Van't Hoff analyses, revealing that GNP-DiMan-DC-SIGN/R binding is enthalpy driven with favourable binding Gibbs free energy changes (ΔG°) being enhanced with increasing GNP size. Finally, we show that increasing the GNP size significantly enhances their antiviral potency. Notably, the DiMan coated 27 nm GNP potently and robustly blocks both DC-SIGN and DC-SIGNR mediated pseudo-Ebola virus cellular entry with an EC50 of ∼23 and ∼49 pM, respectively, making it the most potent glycoconjugate inhibitor against DC-SIGN/R-mediated Ebola cellular infections. Our results have established GNP-glycans as a new tool for quantifying MLGI biophysical parameters and revealed that increasing the GNP scaffold size significantly enhances their MLGI affinities and antiviral potencies.

Quantitative measurement of cation-mediated adhesion of DNA to anionic surfaces.

Anionic polyelectrolytes, such as DNA, are attracted to anionic surfaces in the presence of multivalent cations. A major barrier toward molecular-level understanding of these attractive interactions is the paucity of measurements of the binding strength. Here, atomic force microscopy-based single molecule force spectroscopy was used to quantify the binding free energy of double-stranded DNA to an anionic surface, with complementary density functional theory calculations of the binding energies of metal ion-ligand complexes. The results support both electrostatic attraction and ion-specific binding. Our study suggests that the correlated interactions between counterions are responsible for attraction between DNA and an anionic surface, but the strength of this attraction is modulated by the identity of the metal ion. We propose a mechanism in which the strength of metal-ligand binding, as well as the preference for particular binding sites, influence both the concentration dependence and the strength of the DNA-surface interactions.

Influence of ion and hydration atmospheres on RNA structure and dynamics: insights from advanced theoretical and computational methods.

RNA, a highly charged biopolymer composed of negatively charged phosphate groups, defies electrostatic repulsion to adopt well-defined, compact structures. Hence, the presence of positively charged metal ions is crucial not only for RNA's charge neutralization, but they also coherently decorate the ion atmosphere of RNA to stabilize its compact fold. This feature article elucidates various modes of close RNA-ion interactions, with a special emphasis on Mg2+ as an outer-sphere and inner-sphere ion. Through examples, we highlight how inner-sphere chelated Mg2+ stabilizes RNA pseudoknots, while outer-sphere ions can also exert long-range electrostatic interactions, inducing groove narrowing, coaxial helical stacking, and RNA ring formation. In addition to investigating the RNA's ion environment, we note that the RNA's hydration environment is relatively underexplored. Our study delves into its profound interplay with the structural dynamics of RNA, employing state-of-the-art atomistic simulation techniques. Through examples, we illustrate how specific ions and water molecules are associated with RNA functions, leveraging atomistic simulations to identify preferential ion binding and hydration sites. However, understanding their impact(s) on the RNA structure remains challenging due to the involvement of large length and long time scales associated with RNA's dynamic nature. Nevertheless, our contributions and recent advances in coarse-grained simulation techniques offer insights into large-scale structural changes dynamically linked to the RNA ion atmosphere. In this connection, we also review how different cutting-edge computational simulation methods provide a microscopic lens into the influence of ions and hydration on RNA structure and dynamics, elucidating distinct ion atmospheric components and specific hydration layers and their individual and collective impacts.

Excitation generated preferential binding sites for ethane on porous carbon-copper porphyrin sorbents: ethane/ethylene adsorptive separation improved by light.

Energy-efficient separation of C2H6/C2H4 is a great challenge, for which adsorptive separation is very promising. C2H6-selective adsorption has big implications, while the design of C2H6-sorbents with ideal adsorption capability, particularly with the C2H6/C2H4-selectivity exceeded 2.0, is still challenging. Instead of the current strategies such as chemical modification or pore space modulation, we propose a new methodology for the design of C2H6-sorbents. With a Cu-TCPP [TCPP = 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin] framework dispersed onto a microporous carbon and a hierarchical-pore carbon, two composite sorbents are fabricated. The composite sorbents exhibit enhanced C2H6-selective adsorption capabilities with visible light, particularly the composite sorbent based on the hierarchical-pore carbon, whose C2H6 and C2H4 adsorption capacities (0 °C, 1 bar) are targetedly increased by 27% and only 1.8% with visible light, and therefore, an C2H6-selectivity (C2H6/C2H4 = 10/90, v/v) of 4.8 can be realized. With visible light, the adsorption force of the C2H6 molecule can be asymmetrically enhanced by the excitation enriched electron density over the adsorption sites formed via the close interaction between the Cu-TCPP and the carbon layer, whereas that of the C2H4 molecule is symmetrically altered and the forces cancelled each other out. This strategy may open up a new route for energy-efficient adsorptive separation of C2H6/C2H4 with light.

An in silico investigation on the binding site preference of PD-1 and PD-L1 for designing antibodies for targeted cancer therapy.

Cancer control and treatment remain a significant challenge in cancer therapy and recently immune checkpoints has considered as a novel treatment strategy to develop anti-cancer drugs. Many cancer types use the immune checkpoints and its ligand, PD-1/PD-L1 pathway, to evade detection and destruction by the immune system, which is associated with altered effector function of PD-1 and PD-L1 overexpression on cancer cells to deactivate T cells. In recent years, mAbs have been employed to block immune checkpoints, therefore normalization of the anti-tumor response has enabled the scientists to develop novel biopharmaceuticals. In vivo affinity maturation of antibodies in targeted therapy has sometimes failed, and current experimental methods cannot accommodate the accurate structural details of protein-protein interactions. Therefore, determining favorable binding sites on the protein surface for modulator design of these interactions is a major challenge. In this study, we used the in silico methods to identify favorable binding sites on the PD-1 and PD-L1 and to optimize mAb variants on a large scale. At first, all the binding areas on PD-1 and PD-L1 have been identified. Then, using the RosettaDesign protocol, thousands of antibodies have been generated for 11 different regions on PD-1 and PD-L1 and then the designs with higher stability, affinity, and shape complementarity were selected. Next, molecular dynamics simulations and MM-PBSA analysis were employed to understand the dynamic, structural features of the complexes and measure the binding affinity of the final designs. Our results suggest that binding sites 1, 3 and 6 on PD-1 and binding sites 9 and 11 on PD-L1 can be regarded as the most appropriate sites for the inhibition of PD-1-PD-L1 interaction by the designed antibodies. This study provides comprehensive information regarding the potential binding epitopes on PD-1 which could be considered as hotspots for designing potential biopharmaceuticals. We also showed that mutations in the CDRs regions will rearrange the interaction pattern between the designed antibodies and targets (PD-1 and PD-L1) with improved affinity to effectively inhibit protein-protein interaction and block the immune checkpoint.

Viral peptide conjugates for metal-warhead delivery to chromatin.

The presence of heavy metal groups can endow compounds with unique structural and chemical attributes beneficial for developing highly potent therapeutic agents and effective molecular labels. However, metallocompound binding site specificity is a major challenge that dictates the level of off-site targeting, which is a limiting factor in finding safer and more effective metal-based drugs. Here we designed and tested a family of metallopeptide conjugates based on two different chromatin-tethering viral proteins and a drug being repurposed for cancer, the Au(i) anti-arthritic auranofin. The viral peptides associate with the acidic patch of the nucleosome while the gold moiety can bind allosterically to the H3 H113 imidazole. To achieve synthesis of the conjugates, we also engineered a sulfur-free, nucleosome-binding Kaposi's sarcoma herpesvirus LANA peptide with a methionine-to-ornithine substitution and coupled the peptide to the metal group in a final step using click chemistry. The four conjugates tested are all selectively cytotoxic towards tumor cell lines, but the choice of viral peptide and mode of linkage to the Au(i) group influences metal binding site preference. Our findings suggest that viral peptide-metalloconjugates have potential for use in chromatin delivery of therapeutic warheads and as nucleosome-specific tags.

Aromatic vs alicyclic: Hydrophobicity of the ionic liquid on protein stability and fibril formation

Ionic liquids (ILs) are environment-friendly solvents with potential applications in biochemical reactions and as pharmaceutical ingredients. They are used to stabilize proteins and act as anti-aggregation agents in which the hydrophobic effect of ILs is suggested to have a primary role. To understand the differences in the effect of aromatic and alicyclic cations of ILs on thermal stability and fibrillation propensity of proteins, the effect of two alicyclic (butyl-1-methylpyrrolidinium and butyl-1-methylpiperidinium) and two aromatic (butyl-4-methylpyridinium and butyl-3-methylimidazolium) ILs on lysozyme was investigated by spectroscopy, densitometry, and molecular dynamics simulation. The results suggest that all the ILs switch the fibrillation pathway from nucleation-independent to nucleation-dependent mechanism at ≥200 mM. The fibrillation is slowed down by 28–35-fold in 500 mM of ILs, exceptionally pyrrolidinium-IL delays the fibrillation by ∼70-fold. All the ILs destabilize the protein during thermal denaturation. Though in all cases the unfolding is enthalpy-driven, pyridinium-IL at higher concentrations exhibits an entropy-driven unfolding effect. The volumetric analysis shows a higher thermal expansion coefficient for Lyz in pyridinium-IL which might be due to the increased internal cavity volume of the protein upon binding of the IL. These results corroborate with the computational studies that the preferential interaction coefficient of aromatic-ILs is more, particularly at a higher concentration (200 mM). Four preferable binding sites for ILs on the protein could be identified that includes the amyloidogenic regions, β-hairpin and the hydrophobic cluster 5. Also, the ILs are found to preferably interact with the protein through their alicyclic/aromatic ring and alkyl sidechain. These results suggest that the binding of ILs to the amyloidogenic region inhibits the fibril formation which has a similar effect for both alicyclic and aromatic ILs whereas aromatic-ILs show a larger destabilization effect than alicyclic-ILs at higher concentrations.

Probing the interaction of lysozyme with cardiac glycoside digitoxin: experimental and in silico analyses.

Digitoxin is a cardiac glycoside used to treat heart failure and heart arrhythmia. However, its therapeutic concentration range is very narrow. High doses of digitoxin are associated with severe side effects; therefore, it is necessary to develop the delivery system which can control the plasma levels of it. In this context, the binding of lysozyme, an important protein having many applications, with digitoxin has been studied to see the ability of the former as a carrier. The studies were carried out using both experimental and computational methods. The intrinsic fluorescence of lysozyme increased on the addition of digitoxin. Fluorescence results suggested that there was a strong interaction between lysozyme and digitoxin which was favored, mainly, by hydrophobic forces. Further, digitoxin affected the secondary structure of lysozyme slightly by causing the partial unfolding of lysozyme. The preferred binding site of digitoxin within lysozyme was the large cavity of the protein. Molecular docking studies also established the principal role of hydrophobic forces in the binding with a significant support of hydrogen bonding. Frontier molecular orbitals of free digitoxin and in complexation with lysozyme were also computed and discussed. The findings from molecular dynamics simulation studies elucidate that, when contrasted with the first and third conformations of the digitoxin-bound lysozyme complex, the second conformation promotes structural stability, reduces flexibility, and enhances the compactness and folding properties of lysozyme. The overall study shows that lysozyme could act as a potential carrier for digitoxin in pharmaceutical formulations.

CCRG-05. A UNIFYING AND SELF-REINFORCING MECHANISM OF TUMOR IMMORTALITY IN TERT PROMOTER MUTANT CANCERS

Abstract Telomerase Reverse Transcriptase promoter mutations enable tumor cell immortality in millions of cancer patients annually. While prior therapies targeting telomerase lacked tumor selectivity and were poorly tolerated, TERT promoter mutations and their regulation present a unique opportunity for tumor specific reversal of cellular immortality. The two hotspot mutations, G228A and G250A, generate identical de novo E26 transformation specific (ETS) transcription factor binding sites. However, the 28 ETS factors share a similar binding site preference, raising the question of whether all ETS factors can reactivate TERT. In glioblastoma and a few other cancers, we have shown the de novo ETS site along with a nearby native ETS site (ETS-195/200) recruit one ETS factor, the GA-binding protein (GABP) tetramer, to reactivate TERT. This GABP-mediated mechanism may be restricted to specific cancer types and the two hotspot mutations or could be widely relevant to any cancer with a de novo ETS TERT promoter mutation. We have found that the GABP tetramer activates 6 distinct mutant TERT promoters across 16 different cancer types. However, TERT expression is maintained in most tetramer depleted tumor cells as the tetramer is serially replaced by GABP dimers and weakly by a paralogous tetramer complex that increase following disengagement of GABP tetramer-mediated negative feedback loops. Elimination of both the tetramer and dimer reinstates epigenetic repression of TERT, shortens telomeres, and prevents cancer cell division. However, some knockout cancer cells avoid this fate through weak activation of TERT by the paralogous GABP tetramer complex. The compensatory maintenance of TERT expression can be overcome with a dominant negative GABPB1, leading to TERT silencing, evidenced by gene expression and non-invasive metabolic imaging correlates, and telomere shortening in TERT promoter mutant glioblastoma. We are currently investigating the delivery of this dominant negative to tumor cells via clinically approved retroviral replicating vectors.

Shape-Induced Variations in Aromatic Thiols Adsorption on Gold Nanoparticle: A Novel Method for Accurate Evaluation of Adsorbed Molecules.

Nonspherical gold nanoparticles (GNPs) are increasingly used to enhance sensitivity and selectivity in analytical methods such as surface-enhanced Raman spectroscopy (SERS) for detecting trace biomarkers. However, there is limited research on the adsorption properties of aromatic thiols onto gold nanoparticles of different morphologies, where surface curvature varies significantly at the molecular level. In this study, we investigated the adsorption kinetics of 4-mercaptobenzoic acid, an aromatic molecule, on GNPs with different shapes using SERS. Our findings revealed significant differences in the adsorption behavior and binding site preferences of aromatic thiols on GNPs with distinct morphologies. While thiol molecules consider any surface site on nanospheres equally appealing, nanostars exhibit variations in curvature and surface energy, leading to initial binding with further repositioning from the tips of the nanostar after plasmon activation. To address these differences, we proposed a universal method to evaluate the quantity of tightly bound adsorbed molecules on GNPs independently of the particle size, shape, or concentration.

Synthesis of amine functionalized SiO2 microspheres loaded polymeric hydrogel and its application for simultaneous eviction of divalent metal ions (Pb2+, Cd2+, Cu2+ and Ni2+)

Recovery of divalent cationic elements from aquatic system is deliberated as reconciliation of rising industrial growth and access to quality drinking water. Herein we report tactically designed amino functionalized silica core-shell nanoparticle loaded polymeric hydrogel network as benchmark sorbent for divalent metal ions (Pb2+, Cd2+, Cu2+, Ni2+). The simple fabrication method for silica core-shell microspheres involves sol-gel synthesis of the polystyrene (PS) core, afterwards the formation of the silica shell by hydrolysis and condensation reaction, and finally the dissolution of the PS core. The surface of synthesized SiO2 microspheres was amine functionalized before being impregnated within a alginate matrix of polymer to generate amino functionalized SiO2 microsphere loaded polymeric hydrogel [NH2–SiO2MS-Ca-Alg] beads. Combining functionalized silica into polymeric hydrogel structure resulted extraordinary sorption capacities for divalent metal ions [191.4, 70.7, 64.9 and 35.8 mg g−1 respectively for Pb, Cd, Cu and Ni]. Density functional theory calculations were carried out to find out binding energies, preferred binding site of all four studied ions and structural changes due to their sorption. Structural evidence derived from experimental observations corroborate with the DFT analysis and speciation diagram of the divalent metal ion species. Plausible mechanistic pathway and differential uptake of four divalent metal ions were explained based on both experimental findings and theoretical studies. Developed NH2–SiO2 MS-Ca-Alg beads offered outstanding selectivity towards four divalent cations in a single prescription, excellent employability under ambient conditions without affecting essential water quality parameters which are significant breakthroughs for sustainable water remediation applications.

DMD antisense oligonucleotide mediated exon skipping efficiency correlates with flanking intron retention time and target position within the exon

ABSTRACT Mutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic approach that allows production of a shorter but functional protein. As DMD causing mutations can affect most of the 79 exons encoding dystrophin, a wide variety of AONs are needed to treat the patient population. Design of AONs is largely guided by trial-and-error, and it is yet unclear what defines the skippability of an exon. Here, we use a library of phosphorodiamidate morpholino oligomer (PMOs) AONs of similar physical properties to test the skippability of a large number of DMD exons. The DMD transcript is non-sequentially spliced, meaning that certain introns are retained longer in the transcript than downstream introns. We tested whether the relative intron retention time has a significant effect on AON efficiency, and found that targeting an out-of-frame exon flanked at its 5’-end by an intron that is retained in the transcript longer (‘slow’ intron) leads to overall higher exon skipping efficiency than when the 5’-end flanking intron is ‘fast’. Regardless of splicing speed of flanking introns, we find that positioning an AON closer to the 5’-end of the target exon leads to higher exon skipping efficiency opposed to targeting an exons 3’-end. The data enclosed herein can be of use to guide future target selection and preferential AON binding sites for both DMD and other disease amenable by exon skipping therapies.

Probing the interaction of cephalosporin antibiotic “cefoperazone” with lysozyme using spectroscopic and in silico methods: Effect of paracetamol on binding

The interaction of lysozyme with cefoperazone was studied by means of spectroscopic and computational approaches. The change in the UV–visible spectrum of lysozyme in presence of cefoperazone was an indication of the complex formation between them. Fluorescence spectroscopy suggested that there was a fair interaction between the protein and drug which was taken place via dynamic quenching mechanism and the binding ratio was approximately 1:1. The binding was energetically feasible and principally supported by the hydrophobic forces. CD spectroscopic studies have shown that cefoperazone induced the secondary structure of lysozyme by increasing the α-helical contents of the latter. In silico studies revealed that the large nonpolar cavity was the preferred binding site of cefoperazone within lysozyme and the interaction was taken place mainly through hydrophobic forces with small involvement of hydrogen bonding and electrostatic interactions which is in good agreement with the experimental analyses. Effect of paracetamol was also seen on the binding and it was found that paracetamol had a negative influence on the binding between cefoperazone and lysozyme.

Hidden modes of DNA binding by human nuclear receptors

Human nuclear receptors (NRs) are a superfamily of ligand-responsive transcription factors that have central roles in cellular function. Their malfunction is linked to numerous diseases, and the ability to modulate their activity with synthetic ligands has yielded 16% of all FDA-approved drugs. NRs regulate distinct gene networks, however they often function from genomic sites that lack known binding motifs. Here, to annotate genomic binding sites of known and unexamined NRs more accurately, we use high-throughput SELEX to comprehensively map DNA binding site preferences of all full-length human NRs, in complex with their ligands. Furthermore, to identify non-obvious binding sites buried in DNA–protein interactomes, we develop MinSeq Find, a search algorithm based on the MinTerm concept from electrical engineering and digital systems design. The resulting MinTerm sequence set (MinSeqs) reveal a constellation of binding sites that more effectively annotate NR-binding profiles in cells. MinSeqs also unmask binding sites created or disrupted by 52,106 single-nucleotide polymorphisms associated with human diseases. By implicating druggable NRs as hidden drivers of multiple human diseases, our results not only reveal new biological roles of NRs, but they also provide a resource for drug-repurposing and precision medicine.

Electrochemical NO3- Reduction Catalyzed by Atomically Precise Ag30Pd4 Bimetallic Nanocluster: Synergistic Catalysis or Tandem Catalysis?

Electrochemically converting NO3- compounds into ammonia represents a sustainable route to remove industrial pollutants in wastewater and produce valuable chemicals. Bimetallic nanomaterials usually exhibit better catalytic performance than the monometallic counterparts, yet unveiling the reaction mechanism is extremely challenging. Herein, we report an atomically precise [Ag30Pd4 (C6H9)26](BPh4)2 (Ag30Pd4) nanocluster as a model catalyst toward the electrochemical NO3- reduction reaction (eNO3-RR) to elucidate the different role of the Ag and Pd site and unveil the comprehensive catalytic mechanism. Ag30Pd4 is the homoleptic alkynyl-protected superatom with 2 free electrons, and it has a Ag30Pd4 metal core where 4 Pd atoms are located at the subcenter of the metal core. Furthermore, Ag30Pd4 exhibits excellent performance toward eNO3-RR and robust stability for prolonged operation, and it can achieve the highest Faradaic efficiency of NH3 over 90%. In situ Fourier-transform infrared study revealed that a Ag site plays a more critical role in converting NO3- into NO2-, while the Pd site makes a major contribution to catalyze NO2- into NH3. The bimetallic nanocluster adopts a tandem catalytic mechanism rather than a synergistic catalytic effect in eNO3-RR. Such finding was further confirmed by density functional theory calculations, as they disclosed that Ag is the most preferable binding site for NO3-, which then binds a water molecule to release NO2-. Subsequently, NO2- can transfer to the vicinal exposed Pd site to promote NH3 formation.

Relationship between graphene nucleation density and epitaxial growth orientation on Cu(111) surfaces

Crystal domain alignment is required to produce large, single-crystal, two-dimensional materials which are synthesized by epitaxial growth, but it has been unclear what is needed to achieve the alignment. It is presumed to be possible when the symmetries of the two-dimensional material and substrate are consistent. However, experiments show that although graphene and the Cu(111) surface have the same symmetry, graphene domains grown on Cu(111) are not always well aligned. We developed a physical model and used first principles calculations to explain these observations. It is found that small hexagonal graphene domains are always misorientated on the Cu(111) substrate because the vertexes of the graphene domains have preferred binding sites. These misorientation angles vary with the domain size, with near-perfect alignment of the domains on the substrate only being achieved when they become hundreds of nanometers in size. Our results indicate that low nucleation density of graphene on Cu(111) surfaces is necessary to achieve epitaxial alignment of graphene domains.