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Yu, Tao, Yuhong Zhu, Zhaojian He i Shi-Jie Chen. "Predicting Molecular Crowding Effects in Ion–RNA Interactions". Journal of Physical Chemistry B 120, nr 34 (12.08.2016): 8837–44. http://dx.doi.org/10.1021/acs.jpcb.6b05625.
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Edwards, Thomas E., i Snorri Th Sigurdsson. "EPR spectroscopic analysis of TAR RNA–metal ion interactions". Biochemical and Biophysical Research Communications 303, nr 2 (kwiecień 2003): 721–25. http://dx.doi.org/10.1016/s0006-291x(03)00411-x.
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Fingerhut, Benjamin P., Eva M. Bruening, Jakob Schauss, Torsten Siebert i Thomas Elsaesser. "Interactions of RNA and Water probed by 2D-IR Spectroscopy". EPJ Web of Conferences 205 (2019): 10003. http://dx.doi.org/10.1051/epjconf/201920510003.
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Combined experimental-theoretical investigation of ultrafast hydration dynamics of an A-form RNA double helix in water reveals an ordered arrangement of water molecules and provides boundary conditions for the ion atmosphere around the polyanionic RNA.
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Edwards, Thomas E., Tamara M. Okonogi i Snorri Th Sigurdsson. "Investigation of RNA-Protein and RNA-Metal Ion Interactions by Electron Paramagnetic Resonance Spectroscopy". Chemistry & Biology 9, nr 6 (czerwiec 2002): 699–706. http://dx.doi.org/10.1016/s1074-5521(02)00150-3.
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Nguyen, Hung T., Naoto Hori i D. Thirumalai. "Theory and simulations for RNA folding in mixtures of monovalent and divalent cations". Proceedings of the National Academy of Sciences 116, nr 42 (30.09.2019): 21022–30. http://dx.doi.org/10.1073/pnas.1911632116.
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RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion–phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.
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Le, Shu-Yun, Jih-H. Chen, N. Pattabiraman i Jacob V. Maizel. "Ion-RNA Interactions in the RNA Pseudoknot of a Ribosomal Frameshifting Site: Molecular Modeling Studies". Journal of Biomolecular Structure and Dynamics 16, nr 1 (sierpień 1998): 1–11. http://dx.doi.org/10.1080/07391102.1998.10508221.
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Wu, Yuan-Yan, Zhong-Liang Zhang, Jin-Si Zhang, Xiao-Long Zhu i Zhi-Jie Tan. "Multivalent ion-mediated nucleic acid helix-helix interactions: RNA versus DNA". Nucleic Acids Research 43, nr 12 (27.05.2015): 6156–65. http://dx.doi.org/10.1093/nar/gkv570.
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Khan, Mateen A. "Analysis of Ion and pH Effects on Iron Response Element (IRE) and mRNA-Iron Regulatory Protein (IRP1) Interactions". Current Chemical Biology 14, nr 2 (19.11.2020): 88–99. http://dx.doi.org/10.2174/2212796814999200604121937.
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Background: Cellular iron uptake, utilization, and storage are tightly controlled through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism. The interaction of iron regulatory proteins with mRNAs containing an iron responsive element plays a central role in this regulation. The IRE RNA family of mRNA regulatory structures combines absolutely conserved protein binding sites with phylogenetically conserved base pairs that are specific to each IREs and influence RNA/protein stability. Our previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present study is to gain further insight into the differences in protein/RNA stability as a function of pH and ionic strength. Objective: To determine the extent to which the binding affinity and stability of protein/RNA complex was affected by ionic strength and pH. Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction. Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule. The binding affinity of two IRE RNA/IRP was significantly changed with the change in pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6. Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength, with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming that most of the tryptophan residues contribute to protein fluorescence. Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex is destabilized by the change in pH and ionic strength. These observations suggest that both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex formation.
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Lemkul, Justin A. "Same fold, different properties: polarizable molecular dynamics simulations of telomeric and TERRA G-quadruplexes". Nucleic Acids Research 48, nr 2 (6.12.2019): 561–75. http://dx.doi.org/10.1093/nar/gkz1154.
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Abstract DNA and RNA sequences rich in guanine can fold into noncanonical structures called G-quadruplexes (GQs), which exhibit a common stem structure of Hoogsteen hydrogen-bonded guanine tetrads and diverse loop structures. GQ sequence motifs are overrepresented in promoters, origins of replication, telomeres, and untranslated regions in mRNA, suggesting roles in modulating gene expression and preserving genomic integrity. Given these roles and unique aspects of different structures, GQs are attractive targets for drug design, but greater insight into GQ folding pathways and the interactions stabilizing them is required. Here, we performed molecular dynamics simulations to study two bimolecular GQs, a telomeric DNA GQ and the analogous telomeric repeat-containing RNA (TERRA) GQ. We applied the Drude polarizable force field, which we show outperforms the additive CHARMM36 force field in both ion retention and maintenance of the GQ folds. The polarizable simulations reveal that the GQs bind bulk K+ ions differently, and that the TERRA GQ accumulates more K+ ions, suggesting different ion interactions stabilize these structures. Nucleobase dipole moments vary as a function of position and also contribute to ion binding. Finally, we show that the TERRA GQ is more sensitive than the telomeric DNA GQ to water-mediated modulation of ion-induced dipole-dipole interactions.
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Kleiman, Diego E., Nawavi Naleem i Serdal Kirmizialtin. "Exploring the Ion-Mediated RNA Interactions of a Helix-Junction-Helix RNA Model through Well-Tempered Metadynamics Simulations". Biophysical Journal 118, nr 3 (luty 2020): 68a—69a. http://dx.doi.org/10.1016/j.bpj.2019.11.549.
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Kumar, Anil. "Ionic interactions in aqueous mixtures of hydrophilic and -phobic ions". Pure and Applied Chemistry 80, nr 6 (1.01.2008): 1267–79. http://dx.doi.org/10.1351/pac200880061267.
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Ions of both hydrophilic and -phobic nature play a pivotal role in biological systems and during synthesis of many organic molecules. However, the interaction of a hydrophilic ion with a hydrophobic one is not a simple physical process and is useful in understanding the rate enhancement in organic transformations and thermal stability of DNA and RNA. During the talk, an attempt was made to present some of the experimental work on aqueous mixtures of these ions carried out at National Chemical Laboratory, Pune, and give a theoretical interpretation of the binary, ternary, and quaternary interactions operating in these systems. The contribution of these interaction terms to the excess free energy of mixing, ΔmGE, will be analyzed.
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Trachman, Robert J., i Adrian R. Ferré-D'Amaré. "An uncommon [K+(Mg2+)2] metal ion triad imparts stability and selectivity to the Guanidine-I riboswitch". RNA 27, nr 10 (13.07.2021): 1257–64. http://dx.doi.org/10.1261/rna.078824.121.
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The widespread ykkC-I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the X-ray crystal structure of the Burkholderia sp. TJI49 ykkC-I subtype-1 (Guanidine-I) riboswitch aptamer domain. Differing from the previously reported structures of orthologs from Dickeya dadantii and Sulfobacillus acidophilus, our Burkholderia structure reveals a chelated K+ ion adjacent to two Mg2+ ions in the guanidine-binding pocket. Thermal melting analysis shows that K+ chelation, which induces localized conformational changes in the binding pocket, improves guanidinium-RNA interactions. Analysis of ribosome structures suggests that the [K+(Mg2+)2] ion triad is uncommon. It is, however, reminiscent of metal ion clusters found in the active sites of ribozymes and DNA polymerases. Previous structural characterization of ykkC-I subtype-2 RNAs, which bind the effector ligands ppGpp and PRPP, indicate that in those paralogs, an adenine responsible for K+ chelation in the Burkholderia Guanidine-I riboswitch is replaced by a pyrimidine. This mutation results in a water molecule and Mg2+ ion binding in place of the K+ ion. Thus, our structural analysis demonstrates how ion and solvent chelation tune divergent ligand specificity and affinity among ykkC-I riboswitches.
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Szulc, Natalia A., Zuzanna Mackiewicz, Janusz M. Bujnicki i Filip Stefaniak. "fingeRNAt—A novel tool for high-throughput analysis of nucleic acid-ligand interactions". PLOS Computational Biology 18, nr 6 (2.06.2022): e1009783. http://dx.doi.org/10.1371/journal.pcbi.1009783.
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Computational methods play a pivotal role in drug discovery and are widely applied in virtual screening, structure optimization, and compound activity profiling. Over the last decades, almost all the attention in medicinal chemistry has been directed to protein-ligand binding, and computational tools have been created with this target in mind. With novel discoveries of functional RNAs and their possible applications, RNAs have gained considerable attention as potential drug targets. However, the availability of bioinformatics tools for nucleic acids is limited. Here, we introduce fingeRNAt—a software tool for detecting non-covalent interactions formed in complexes of nucleic acids with ligands. The program detects nine types of interactions: (i) hydrogen and (ii) halogen bonds, (iii) cation-anion, (iv) pi-cation, (v) pi-anion, (vi) pi-stacking, (vii) inorganic ion-mediated, (viii) water-mediated, and (ix) lipophilic interactions. However, the scope of detected interactions can be easily expanded using a simple plugin system. In addition, detected interactions can be visualized using the associated PyMOL plugin, which facilitates the analysis of medium-throughput molecular complexes. Interactions are also encoded and stored as a bioinformatics-friendly Structural Interaction Fingerprint (SIFt)—a binary string where the respective bit in the fingerprint is set to 1 if a particular interaction is present and to 0 otherwise. This output format, in turn, enables high-throughput analysis of interaction data using data analysis techniques. We present applications of fingeRNAt-generated interaction fingerprints for visual and computational analysis of RNA-ligand complexes, including analysis of interactions formed in experimentally determined RNA-small molecule ligand complexes deposited in the Protein Data Bank. We propose interaction fingerprint-based similarity as an alternative measure to RMSD to recapitulate complexes with similar interactions but different folding. We present an application of interaction fingerprints for the clustering of molecular complexes. This approach can be used to group ligands that form similar binding networks and thus have similar biological properties. The fingeRNAt software is freely available at https://github.com/n-szulc/fingeRNAt.
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Liu, Chuanliang, Ljiljana Paša Tolić, Steven A. Hofstadler, Amy C. Harms, Richard D. Smith, ChulHee Kang i Nanda Sinha. "Probing RegA/RNA Interactions Using Electrospray Ionization–Fourier Transform Ion Cyclotron Resonance–Mass Spectrometry". Analytical Biochemistry 262, nr 1 (sierpień 1998): 67–76. http://dx.doi.org/10.1006/abio.1998.2753.
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Etzion-Fuchs, Anat, David A. Todd i Mona Singh. "dSPRINT: predicting DNA, RNA, ion, peptide and small molecule interaction sites within protein domains". Nucleic Acids Research 49, nr 13 (17.05.2021): e78-e78. http://dx.doi.org/10.1093/nar/gkab356.
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Abstract Domains are instrumental in facilitating protein interactions with DNA, RNA, small molecules, ions and peptides. Identifying ligand-binding domains within sequences is a critical step in protein function annotation, and the ligand-binding properties of proteins are frequently analyzed based upon whether they contain one of these domains. To date, however, knowledge of whether and how protein domains interact with ligands has been limited to domains that have been observed in co-crystal structures; this leaves approximately two-thirds of human protein domain families uncharacterized with respect to whether and how they bind DNA, RNA, small molecules, ions and peptides. To fill this gap, we introduce dSPRINT, a novel ensemble machine learning method for predicting whether a domain binds DNA, RNA, small molecules, ions or peptides, along with the positions within it that participate in these types of interactions. In stringent cross-validation testing, we demonstrate that dSPRINT has an excellent performance in uncovering ligand-binding positions and domains. We also apply dSPRINT to newly characterize the molecular functions of domains of unknown function. dSPRINT’s predictions can be transferred from domains to sequences, enabling predictions about the ligand-binding properties of 95% of human genes. The dSPRINT framework and its predictions for 6503 human protein domains are freely available at http://protdomain.princeton.edu/dsprint.
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Gräf, S., R. Przybilski, G. Steger i C. Hammann. "A database search for hammerhead ribozyme motifs". Biochemical Society Transactions 33, nr 3 (1.06.2005): 477–78. http://dx.doi.org/10.1042/bst0330477.
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The hammerhead ribozyme is the smallest naturally occurring RNA endonuclease. It is found in subviral plant pathogens and transcripts of satellite DNA from a limited number of organisms. We have performed a database search for novel examples of this catalytic RNA, taking into consideration the recently defined structural requirements for an efficient cleavage under physiological magnesium ion concentrations. In this search, we find, apart from the known examples, several hundreds of motifs in organisms of all kingdoms of life. In a first set of experiments, we analysed hammerhead ribozymes from Arabidopsis thaliana. We found that these sequences are tissue-specifically expressed and that they undergo self-cleavage in planta. Furthermore, their activity under physiological magnesium ion concentrations depends on functional loop–loop interactions, as shown by the lack of activity of appropriate mutants.
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Fingerhut, Benjamin P., Jakob Schauss, Achintya Kundu i Thomas Elsaesser. "Aqueous Contact Ion Pairs of Phosphate Groups with Na+, Ca2+ and Mg2+ – Structural Discrimination by Femtosecond Infrared Spectroscopy and Molecular Dynamics Simulations". Zeitschrift für Physikalische Chemie 234, nr 7-9 (27.08.2020): 1453–74. http://dx.doi.org/10.1515/zpch-2020-1614.
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AbstractThe extent of contact and solvent shared ion pairs of phosphate groups with Na+, Ca2+ and Mg2+ ions in aqueous environment and their relevance for the stability of polyanionic DNA and RNA structures is highly debated. Employing the asymmetric phosphate stretching vibration of dimethyl phosphate (DMP), a model system of the sugar-phosphate backbone of DNA and RNA, we present linear infrared, femtosecond infrared pump-probe and absorptive 2D-IR spectra that report on contact ion pair formation via the presence of blue shifted spectral signatures. Compared to the linear infrared spectra, the nonlinear spectra reveal contact ion pairs with increased sensitivity because the spectra accentuate differences in peak frequency, transition dipole moment strength, and excited state lifetime. The experimental results are corroborated by long time scale MD simulations, benchmarked by density functional simulations on phosphate-ion-water clusters. The microscopic interpretation reveals subtle structural differences of ion pairs formed by the phosphate group and the ions Na+, Ca2+ and Mg2+. Intricate properties of the solvation shell around the phosphate group and the ion are essential to explain the experimental observations. The present work addresses a challenging to probe topic with the help of a model system and establishes new experimental data of contact ion pair formation, thereby underlining the potential of nonlinear 2D-IR spectroscopy as an analytical probe of phosphate-ion interactions in complex biological systems.
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Buskiewicz, Iwona, Malgorzata Giel-Pietraszuk, Piotr Mucha, Piotr Rekowski, Gotfryd Kupryszewski i Miroslawa Z. Barciszewska. "Interaction of HIV Tat Peptides With tRNAPhe from Yeast". Collection of Czechoslovak Chemical Communications 63, nr 6 (1998): 842–50. http://dx.doi.org/10.1135/cccc19980842.
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We present data on the interaction of arginine-rich peptides of human immunodeficiency virus (HIV-Tat) with tRNAPhe of Saccharomyces cerevisiae. We have found that tRNA forms complexes with the Tat1 peptide of amino acid sequence GRKKRRQRRRA and its mutants where R is replaced by D-arginine, citrulline or ornithine. The structure of tRNA-Tat1 complex was probed by specific RNases digestions and Pb2+-induced cleavage of phosphodiester bond of guanosine. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe binds to Tat peptide and therefore is specifically protected against RNases and is not hydrolyzed by Pb2+ ion. It seems that the peptide-RNA complex formation depends on direct recognition of guanine moieties of tRNA with arginine residues. These interactions are similar to those observed in many DNA-protein complexes, but are different from those previously observed for TAR RNA-Tat complexes.
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Xu, Xiaochen, Michaela Egger, Hao Chen, Karolina Bartosik, Ronald Micura i Aiming Ren. "Insights into xanthine riboswitch structure and metal ion-mediated ligand recognition". Nucleic Acids Research 49, nr 12 (14.06.2021): 7139–53. http://dx.doi.org/10.1093/nar/gkab486.
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Abstract Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson–Crick base pair to junction J2. Xanthine inserts between a G–U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.
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Huang, Lin, Jia Wang, Andrew M. Watkins, Rhiju Das i David M. J. Lilley. "Structure and ligand binding of the glutamine-II riboswitch". Nucleic Acids Research 47, nr 14 (19.06.2019): 7666–75. http://dx.doi.org/10.1093/nar/gkz539.
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Abstract We have determined the structure of the glutamine-II riboswitch ligand binding domain using X-ray crystallography. The structure was solved using a novel combination of homology modeling and molecular replacement. The structure comprises three coaxial helical domains, the central one of which is a pseudoknot with partial triplex character. The major groove of this helix provides the binding site for L-glutamine, which is extensively hydrogen bonded to the RNA. Atomic mutation of the RNA at the ligand binding site leads to loss of binding shown by isothermal titration calorimetry, explaining the specificity of the riboswitch. A metal ion also plays an important role in ligand binding. This is directly bonded to a glutamine carboxylate oxygen atom, and its remaining inner-sphere water molecules make hydrogen bonding interactions with the RNA.
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Markelova, O. Yu, L. N. Buryanovsky, O. E. Kitam, K. F. Krasnova i A. D. Shved. "Influence of ion interactions on the conformation of ribozyme specific to HIV-1 tat-RNA in solution". Biopolymers and Cell 18, nr 5 (20.09.2002): 452–54. http://dx.doi.org/10.7124/bc.000627.
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Liu, Bin, Yuhong Zuo i Thomas A. Steitz. "Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism". Proceedings of the National Academy of Sciences 113, nr 15 (28.03.2016): 4051–56. http://dx.doi.org/10.1073/pnas.1520555113.
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In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3′-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E. coli transcription initiation complexes (TICs) containing the stress-responsive σS factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σS-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σS factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the −10 element. In addition, σS-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σS-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.
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Lee, Ji-Hye, Intekhab Alam, Kang Rok Han, Sunyoung Cho, Sungho Shin, Seokha Kang, Jai Myung Yang i Kyung Hyun Kim. "Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase". Journal of General Virology 92, nr 7 (1.07.2011): 1607–16. http://dx.doi.org/10.1099/vir.0.031104-0.
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Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.
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Kaye, Nicholas M., Nathan H. Zahler, Eric L. Christian i Michael E. Harris. "Conservation of Helical Structure Contributes to Functional Metal Ion Interactions in the Catalytic Domain of Ribonuclease P RNA". Journal of Molecular Biology 324, nr 3 (listopad 2002): 429–42. http://dx.doi.org/10.1016/s0022-2836(02)01094-x.
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Sun, Yunhao, Limin Qiu, Jinjin Chen, Yao Wang, Jun Qian, Lirong Huang i Haitao Ma. "Construction of circRNA-Associated ceRNA Network Reveals Novel Biomarkers for Esophageal Cancer". Computational and Mathematical Methods in Medicine 2020 (28.08.2020): 1–12. http://dx.doi.org/10.1155/2020/7958362.
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Objective. Esophageal cancer (ESCC) is reported to be the eighth most common malignant tumors worldwide with high mortality. However, the functions of majority circRNAs in ESCC requires to be further explored. Methods. This study identified differently expressed circRNAs in 3 paired ESCC using RNA-sequencing method. The interactions among circRNAs, miRNAs, and mRNAs were predicted using bioinformatics analysis. Results. In this study, using RNA-sequencing method and integrated bioinformatics analysis, 418 overexpressed circRNAs and 637 reduced circRNAs in ESCC sample were identified. Based on the mechanism that circRNAs could play as ceRNAs to modulate targets expression, circRNA-miRNA and circRNA-miRNA-mRNA networks were constructed in this study. Based on the network analysis, 7 circRNAs, including circ_0002255, circ_0000530, circ_0001904, circ_0001005, circ_0000513, circ_0000075, and circ_0001121, were identified as key circRNAs in ESCC. We found that circ_0002255 was related to the regulation of substrate adhesion-dependent cell spreading. circ_0001121 was involved in regulating nucleocytoplasmic transport. circ_0000513 played a key role in regulating Adherens junction, B cell receptor signaling pathway. Meanwhile, we observed circ_0000075 was involved in regulating zinc II ion transport, transition metal ion homeostasis, and angiogenesis. Conclusion. We thought this study could provide novel biomarkers for the prognosis of ESCC.
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Bonneau, Eric, i Pascale Legault. "NMR Localization of Divalent Cations at the Active Site of theNeurosporaVS Ribozyme Provides Insights into RNA–Metal-Ion Interactions". Biochemistry 53, nr 3 (10.01.2014): 579–90. http://dx.doi.org/10.1021/bi401484a.
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Rooj, Arun K., Zhiyong Liu, Carmel M. McNicholas i Catherine M. Fuller. "Physical and functional interactions between a glioma cation channel and integrin-β1 require α-actinin". American Journal of Physiology-Cell Physiology 309, nr 5 (1.09.2015): C308—C319. http://dx.doi.org/10.1152/ajpcell.00036.2015.
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Major plasma membrane components of the tumor cell, ion channels, and integrins play crucial roles in metastasis. Glioma cells express an amiloride-sensitive nonselective cation channel composed of acid-sensing ion channel (ASIC)-1 and epithelial Na+ channel (ENaC) α- and γ-subunits. Inhibition of this channel is associated with reduced cell migration and proliferation. Using the ASIC-1 subunit as a reporter for the channel complex, we found a physical and functional interaction between this channel and integrin-β1. Short hairpin RNA knockdown of integrin-β1 attenuated the amiloride-sensitive current, which was due to loss of surface expression of ASIC-1. In contrast, upregulation of membrane expression of integrin-β1 increased the surface expression of ASIC-1. The link between the amiloride-sensitive channel and integrin-β1 was mediated by α-actinin. Downregulation of α-actinin-1 or -4 attenuated the amiloride-sensitive current. Mutation of the putative binding site for α-actinin on the COOH terminus of ASIC-1 reduced the membrane localization of ASIC-1 and also resulted in attenuation of the amiloride-sensitive current. Our data suggest a novel interaction between the amiloride-sensitive glioma cation channel and integrin-β1, mediated by α-actinin. This interaction may form a mechanism by which channel activity can regulate glioma cell proliferation and migration.
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Mitash, Nilay, Joshua E. Donovan i Agnieszka Swiatecka-Urban. "The Role of MicroRNA in the Airway Surface Liquid Homeostasis". International Journal of Molecular Sciences 21, nr 11 (28.05.2020): 3848. http://dx.doi.org/10.3390/ijms21113848.
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Mucociliary clearance, mediated by a coordinated function of cilia bathing in the airway surface liquid (ASL) on the surface of airway epithelium, protects the host from inhaled pathogens and is an essential component of the innate immunity. ASL is composed of the superficial mucus layer and the deeper periciliary liquid. Ion channels, transporters, and pumps coordinate the transcellular and paracellular movement of ions and water to maintain the ASL volume and mucus hydration. microRNA (miRNA) is a class of non-coding, short single-stranded RNA regulating gene expression by post-transcriptional mechanisms. miRNAs have been increasingly recognized as essential regulators of ion channels and transporters responsible for ASL homeostasis. miRNAs also influence the airway host defense. We summarize the most up-to-date information on the role of miRNAs in ASL homeostasis and host–pathogen interactions in the airway and discuss concepts for miRNA-directed therapy.
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Xu, Jiayang, Qiansi Chen, Pingping Liu, Wei Jia, Zheng Chen i Zicheng Xu. "Integration of mRNA and miRNA Analysis Reveals the Molecular Mechanism Underlying Salt and Alkali Stress Tolerance in Tobacco". International Journal of Molecular Sciences 20, nr 10 (14.05.2019): 2391. http://dx.doi.org/10.3390/ijms20102391.
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Salinity is one of the most severe forms of abiotic stress and affects crop yields worldwide. Plants respond to salinity stress via a sophisticated mechanism at the physiological, transcriptional and metabolic levels. However, the molecular regulatory networks involved in salt and alkali tolerance have not yet been elucidated. We developed an RNA-seq technique to perform mRNA and small RNA (sRNA) sequencing of plants under salt (NaCl) and alkali (NaHCO3) stress in tobacco. Overall, 8064 differentially expressed genes (DEGs) and 33 differentially expressed microRNAs (DE miRNAs) were identified in response to salt and alkali stress. A total of 1578 overlapping DEGs, which exhibit the same expression patterns and are involved in ion channel, aquaporin (AQP) and antioxidant activities, were identified. Furthermore, genes involved in several biological processes, such as “photosynthesis” and “starch and sucrose metabolism,” were specifically enriched under NaHCO3 treatment. We also identified 15 and 22 miRNAs that were differentially expressed in response to NaCl and NaHCO3, respectively. Analysis of inverse correlations between miRNAs and target mRNAs revealed 26 mRNA-miRNA interactions under NaCl treatment and 139 mRNA-miRNA interactions under NaHCO3 treatment. This study provides new insights into the molecular mechanisms underlying the response of tobacco to salinity stress.
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de Assis Filho, F. M., O. R. Paguio, J. L. Sherwood i C. M. Deom. "Symptom induction by Cowpea chlorotic mottle virus on Vigna unguiculata is determined by amino acid residue 151 in the coat protein". Journal of General Virology 83, nr 4 (1.04.2002): 879–83. http://dx.doi.org/10.1099/0022-1317-83-4-879.
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The type strain of Cowpea chlorotic mottle virus (CCMV-T) produces a bright chlorosis in cowpea (Vigna unguiculata cv. California Blackeye). The attenuated variant (CCMV-M) induces mild green mottle symptoms that were previously mapped to RNA 3. Restriction fragment exchanges between RNA 3 cDNA clones of CCMV-T and CCMV-M that generate infectious transcripts and site-directed mutagenesis indicated that the codon encoding amino acid residue 151 of the coat protein determines the symptom phenotypes of CCMV-T and CCMV-M. Amino acid 151 is within an α-helical structure required for calcium ion binding and virus particle stability. No differences in virion stability or accumulation were detected between CCMV-T and CCMV-M. Mutational analysis suggested that the amino acid at position 151 and not the nucleotide sequence induce the symptom phenotype. Thus, it is likely that subtle influences by amino acid residue 151 in coat protein–host interactions result in chlorotic and mild green mottle symptoms.
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Vijayaraghavan, Bhooma, Giri Padmanabhan i Kumaresan Ramanathan. "Assessment of siRNA as a therapeutic molecule in Transient Receptor Potential Channel 5 gene silencing: a computational approach". Biomedical Research and Therapy 5, nr 1 (19.01.2018): 1911–22. http://dx.doi.org/10.15419/bmrat.v5i1.405.
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Background: Ion channels play a crucial role in Glomerular filter damage that contributes to albuminuria. Transient receptor potential channel 5 (TRPC5) gene mediating such damage, demand for its target specific inhibition by RNA interference mechanism. Designing and selecting potential siRNA for TRPC5 gene silencing by computational analysis.
Materials & Methods: The mRNA sequence was retrieved from NCBI (National Center for Biotechnology Information). siRNA sequences were designed specifically from target genes using InvivoGen siRNA wizard software. Thermodynamic RNA-RNA interactions were used to evaluate the gene silencing efficiency by minimum free energy of hybridization; the hybridization structures were also obtained using BIBISERV2-RNAHybrid.
Results: The minimum free energy of hybridization of the three designed siRNAs (siRNA1, siRNA2 and siRNA3) were as follows: -28.2 kcal/mol, -24.1 kcal/mol, and-25.6 kcal/mol. Their corresponding GC content were 47.62%, 52.38% and 47.62%, respectively. Thus, siRNA1 had the least minimum free energy of hybridization (i.e. -28.2 kcal/mol) with low GC content (47.62%), and high linearity with minimal h-b index and loop structure.
Conclusion: RNAi therapy can provide a new platform for efficient and targeted therapeutics. Further in vivo investigations are necessary to further validate their efficacy.
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Liao, Zengwei, Takuji Oyama, Yumi Kitagawa, Katsuo Katayanagi, Kosuke Morikawa i Masayuki Oda. "Pivotal role of a conserved histidine in Escherichia coli ribonuclease HI as proposed by X-ray crystallography". Acta Crystallographica Section D Structural Biology 78, nr 3 (23.02.2022): 390–98. http://dx.doi.org/10.1107/s2059798322000870.
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The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme–Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme–Zn2+ complex a single metal ion was found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.
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Pagliari, Laura, Giulia Tarquini, Alberto Loschi, Sara Buoso, Gregor Kapun, Paolo Ermacora i Rita Musetti. "Gimme shelter: three-dimensional architecture of the endoplasmic reticulum, the replication site of grapevine Pinot gris virus". Functional Plant Biology 48, nr 10 (2021): 1074. http://dx.doi.org/10.1071/fp21084.
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Grapevine leaf mottling and deformation is a novel grapevine disease that has been associated with grapevine Pinot gris virus (GPGV). The virus was observed exclusively inside membrane-bound structures in the bundle sheath cells of the infected grapevines. As reported widely in the literature, many positive-sense single-stranded RNA viruses modify host-cell membranes to form a variety of deformed organelles, which shelter viral genome replication from host antiviral compounds. Morphologically, the GPGV-associated membranous structures resemble the deformed endoplasmic reticulum described in other virus-host interactions. In this study we investigated the GPGV-induced membranous structures observed in the bundle sheath cells of infected plants. The upregulation of different ER stress-related genes was evidenced by RT-qPCR assays, further confirming the involvement of the ER in grapevine/GPGV interaction. Specific labelling of the membranous structures with an antibody against luminal-binding protein identified them as ER. Double-stranded RNA molecules, which are considered intermediates of viral replication, were localised exclusively in the ER-derived structures and indicated that GPGV exploited this organelle to replicate itself in a shelter niche. Novel analyses using focussed ion-beam scanning electron microscopy (FIB-SEM) were performed in grapevine leaf tissues to detail the three-dimensional organisation of the ER-derived structures and their remodelling due to virus replication.
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Ferrero, Diego S., Michela Falqui i Nuria Verdaguer. "Snapshots of a Non-Canonical RdRP in Action". Viruses 13, nr 7 (28.06.2021): 1260. http://dx.doi.org/10.3390/v13071260.
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RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication and transcription. The closed “right hand” architecture of RdRPs encircles seven conserved structural motifs (A to G) that regulate the polymerization activity. The four palm motifs, arranged in the sequential order A to D, are common to all known template dependent polynucleotide polymerases, with motifs A and C containing the catalytic aspartic acid residues. Exceptions to this design have been reported in members of the Permutotetraviridae and Birnaviridae families of positive single stranded (+ss) and double-stranded (ds) RNA viruses, respectively. In these enzymes, motif C is located upstream of motif A, displaying a permuted C–A–B–D connectivity. Here we study the details of the replication elongation process in the non-canonical RdRP of the Thosea asigna virus (TaV), an insect virus from the Permutatetraviridae family. We report the X-ray structures of three replicative complexes of the TaV polymerase obtained with an RNA template-primer in the absence and in the presence of incoming rNTPs. The structures captured different replication events and allowed to define the critical interactions involved in: (i) the positioning of the acceptor base of the template strand, (ii) the positioning of the 3’-OH group of the primer nucleotide during RNA replication and (iii) the recognition and positioning of the incoming nucleotide. Structural comparisons unveiled a closure of the active site on the RNA template-primer binding, before rNTP entry. This conformational rearrangement that also includes the repositioning of the motif A aspartate for the catalytic reaction to take place is maintained on rNTP and metal ion binding and after nucleotide incorporation, before translocation.
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Guo, Shaoke, Mengli Cao, Xingdong Wang, Lin Xiong, Xiaoyun Wu, Pengjia Bao, Min Chu i in. "Changes in Transcriptomic Profiles in Different Reproductive Periods in Yaks". Biology 10, nr 12 (25.11.2021): 1229. http://dx.doi.org/10.3390/biology10121229.
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Yak reproductive characteristics have received extensive attention, though the molecular regulation mechanism of its ovarian activity remains to be explored. Therefore, this study initially conducted a comparative analysis of yak ovarian activities in anestrus, estrus, and pregnancy regarding their morphology and histology, followed by implementing RNA sequencing (RNA-seq) technology to detect the overall gene expression and biological mechanism in different reproductive stages. H&E staining showed that there were more growing follicles and mature follicles in ovarian tissue sections during estrus than ovarian tissues during non-estrus. The RNA-seq analysis of yak ovary tissues in three periods showed that DEGs related to follicular development and hormone metabolism were screened in the three comparison groups, such as COL1A2, NR4A1, THBS2, PTGS2, SCARB1, STAR, and WNT2B. Bioinformatics analysis showed that these DEGs are involved in ion binding, cell development, metabolic processes, enriched in ECM–receptor interactions, steroid biosynthesis, together with aldosterone generation/discharge and Wnt/PI3K-Akt signaling pathways. In addition, we speculate alternate splice development events to have important role/s in regulating ovarian functional genomic expression profiles. These results provide essential knowledge aimed at scrutinizing pivotal biomarkers for yak ovarian activity, together with paving the way for enhancing researchers’ focus on improving yak reproductive performance.
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Peng, Guiqing, Yan Yan, Chengliang Zhu, Shiqun Wang, Xiaohong Yan, Lili Lu, Wei Li i in. "Borna Disease Virus P Protein Affects Neural Transmission through Interactions with Gamma-Aminobutyric Acid Receptor-Associated Protein". Journal of Virology 82, nr 24 (24.09.2008): 12487–97. http://dx.doi.org/10.1128/jvi.00877-08.
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ABSTRACT Borna disease virus (BDV) is one of the infectious agents that causes diseases of the central nervous system in a wide range of vertebrate species and, perhaps, in humans. The phosphoprotein (P) of BDV, an essential cofactor of virus RNA-dependent RNA polymerase, is required for virus replication. In this study, we identified the gamma-aminobutyric acid receptor-associated protein (GABARAP) with functions in neurobiology as one of the viral P protein-interacting cellular factors by using an approach of phage display-based protein-protein interaction analysis. Direct binding between GABARAP and P protein was confirmed by coimmunoprecipitation, protein pull-down, and mammalian two-hybrid analyses. GABARAP originally was identified as a linker between the gamma-aminobutyric acid receptor (GABAR) and the microtubule to regulate receptor trafficking and plays important roles in the regulation of the inhibitory neural transmitter gamma-aminobutyric acid (GABA). We showed that GABARAP colocalizes with P protein in the cells infected with BDV or transfected with the P gene, which resulted in shifting the localization of GABARAP from the cytosol to the nucleus. We further demonstrated that P protein blocks the trafficking of GABAR, a principal GABA-gated ion channel that plays important roles in neural transmission, to the surface of cells infected with BDV or transfected with the P gene. We proposed that during BDV infection, P protein binds to GABARAP, shifts the distribution of GABARAP from the cytoplasm to the nucleus, and disrupts the trafficking of GABARs to the cell membranes, which may result in the inhibition of GABA-induced currents and in the enhancement of hyperactivity and anxiety.
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Andreeva, Tatyana V., Natalya V. Maluchenko, Anastasiia L. Sivkina, Oleg V. Chertkov, Maria E. Valieva, Elena Y. Kotova, Mikhail P. Kirpichnikov, Vasily M. Studitsky i Alexey V. Feofanov. "Na+ and K+ Ions Differently Affect Nucleosome Structure, Stability, and Interactions with Proteins". Microscopy and Microanalysis 28, nr 1 (1.12.2021): 243–53. http://dx.doi.org/10.1017/s1431927621013751.
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Inorganic ions are essential factors stabilizing nucleosome structure; however, many aspects of their effects on DNA transactions in chromatin remain unknown. Here, differential effects of K+ and Na+ on the nucleosome structure, stability, and interactions with protein complex FACT (FAcilitates Chromatin Transcription), poly(ADP-ribose) polymerase 1, and RNA polymerase II were studied using primarily single-particle Förster resonance energy transfer microscopy. The maximal stabilizing effect of K+ on a nucleosome structure was observed at ca. 80–150 mM, and it decreased slightly at 40 mM and considerably at >300 mM. The stabilizing effect of Na+ is noticeably lower than that of K+ and progressively decreases at ion concentrations higher than 40 mM. At 150 mM, Na+ ions support more efficient reorganization of nucleosome structure by poly(ADP-ribose) polymerase 1 and ATP-independent uncoiling of nucleosomal DNA by FACT as compared with K+ ions. In contrast, transcription through a nucleosome is nearly insensitive to K+ or Na+ environment. Taken together, the data indicate that K+ environment is more preserving for chromatin structure during various nucleosome transactions than Na+ environment.
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Neuman, Benjamin W., Jeremiah S. Joseph, Kumar S. Saikatendu, Pedro Serrano, Amarnath Chatterjee, Margaret A. Johnson, Lujian Liao i in. "Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3". Journal of Virology 82, nr 11 (26.03.2008): 5279–94. http://dx.doi.org/10.1128/jvi.02631-07.
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ABSTRACT Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.
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Acharya, Parag, i Jyoti Chattopadhyaya. "Electrostatic cross-modulation of the pseudoaromatic character in single-stranded RNA by nearest-neighbor interactions". Pure and Applied Chemistry 77, nr 1 (1.01.2005): 291–311. http://dx.doi.org/10.1351/pac200577010291.
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The generation of a single anionic or cationic center at an alkaline or acidic pH in a given molecule presents a unique opportunity to examine the electrostatic make-up of these molecules both at the neutral or ionic state. The generation of a single cationic center in the phenyl-nicotinamide system provided new straightforward evidence showing that the charge density of the electron-deficient pyridinium was actually enhanced by the donation of the charge from the electron-rich phenyl group (i.e., the pyridinyl became more basic by ca. 0.5 pKa unit compared to an analogous system where phenyl was absent) owing to the electrostatic interactions between these two moieties. On the other hand, the generation of the 5'-guanylate ion in the hexameric single-strand (ss) RNA [5'-GAAAAC-3'], in comparison with the constituent trimeric, tetrameric, and pentameric-ssRNAs, has unequivocally shown how far the electrostatic cross-talk (as an interplay of Coulombic attractive or repulsive forces) in this electronically coupled system propagates through the intervening pAp nucleotide steps until the terminal pC-3' residue in comparison with the neutral counterpart. The footprint of the propagation of this electrostatic cross-talk among the neighboring nucleobases is evident by measurement of pKas from the marker protons of ionization point (i.e., of G) as well as from the neighboring marker protons (i.e., of A or C) in the vicinity, as well as from the change of the chemical environment (i.e., chemical shifts) around their aromatic marker protons (δH2, δH8, δH5, and δH6) owing to a change of the stacking-destacking equilibrium as a function of pH.
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Hwang, William L., Karthik Jagadeesh, Jimmy Guo, Hannah I. Hoffman, Orr Ashenberg, Eugene Drokhlyansky, Nicholas Van Wittenberghe i in. "Single-nucleus RNA-seq of frozen archival primary pancreatic ductal adenocarcinoma uncovers multi-compartment intratumoral heterogeneity associated with neoadjuvant treatment." Journal of Clinical Oncology 38, nr 15_suppl (20.05.2020): 4633. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.4633.
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4633 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease and existing molecular subtypes do not inform clinical decisions. Previously identified bulk transcriptomic subtypes of PDAC were often unintentionally driven by “contaminating” stroma. RNA extraction from pancreatic tissue is difficult and prior single-cell RNA-seq efforts have been limited by suboptimal dissociation/RNA quality and poor performance in the setting of neoadjuvant treatment. We developed a robust single-nucleus RNA-seq (sNuc-seq) technique compatible with frozen archival PDAC specimens. Methods: Single nuclei suspensions were extracted from frozen primary PDAC specimens (n = 27) derived from patients with (borderline)-resectable PDAC who underwent surgical resection with or without neoadjuvant chemoradiotherapy (CRT). Approximately 170,000 nuclei were processed with the 10x Genomics Single Cell 3’ v3 pipeline and gene expression libraries were sequenced (Illumina HiSeq X). Results: Distinct nuclei clusters with gene expression profiles/inferred copy number variation analysis consistent with neoplastic, acinar, ductal, fibroblast, endothelial, endocrine, lymphocyte, and myeloid populations were identified with proportions similar to corresponding multiplexed ion beam imaging. Non-negative matrix factorization revealed intra-tumoral heterogeneity shared across patients. Neoplastic cells featured eight distinct transcriptional topics characterized by developmental (epithelial, mesenchymal, endoderm progenitor, neural progenitor) and environmental (anabolic, catabolic, cycling, hypoxic) programs. CAFs exhibited four different transcriptional topics (activated/desmoplastic, myofibroblast, neurogenic, osteochondral). Differential gene expression and gene set enrichment analyses demonstrated that CRT was associated with an enrichment in myogenic programs in CAFs, activation pathways in immune cells, and type I/II interferons in malignant cells. CRT was also associated with a depletion in developmental programs within malignant cells. Conclusions: We uncovered significant intratumoral heterogeneity and treatment-associated differences in the malignant, fibroblast, and immune compartments of PDAC using sNuc-seq. Deconvolution of clinically-annotated bulk RNA-seq cohorts and characterization of intercellular interactions with receptor-ligand analysis and spatial transcriptomics are ongoing.
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Wan Othman, Wan N. Nazneem, Fatimah Salim, Nor N. Abdullah, Syahrul I. Abu Bakar, Khalijah Awang, Lalith Jayasinghe i Nor H. Ismail. "(R)-13aα-Densiindolizidine, A New Phenanthroindolizidine Alkaloid From Cryptocarya densiflora Blume (Lauraceae) and Molecular Docking Against SARS-CoV-2". Natural Product Communications 17, nr 8 (sierpień 2022): 1934578X2211142. http://dx.doi.org/10.1177/1934578x221114227.
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Cryptocarya densiflora Blume (Lauraceae) is an evergreen tree widely distributed throughout the hills and mountain forests up to 1500 m in Malaysia and Indonesia. The plant has been reported to contain phenanthroindolizidine-type of alkaloids. In the present work, a new phenanthroindolizidine alkaloid named ( R)-13a α-densiindolizidine, was isolated from the dichloromethane (DCM) extract of the leaves. The structure of the alkaloid was established based on 1D and 2D nuclear magnetic resonance (NMR) and liquid chromatography mass spectrometry-ion trap-time of flight (LCMS-IT-TOF) analysis. ( R)-13a α-densiindolizidine displayed binding interactions with crucial amino acid residues in the active sites ofsevere acute respiratory syndrome coronavirus 2 Mpro (SARS-CoV-2 Mpro) and RNA-dependent protease (RdRp) in silico, whilst fulfilling theabsorption, distribution, metabolism, excretion, and toxicity (ADMET) criteria and Lipinsky's rule, thus revealing its potential as a lead compound.
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Lopez, Melany N., Timothy J. Wilding i James E. Huettner. "Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles". Journal of General Physiology 142, nr 3 (12.08.2013): 225–39. http://dx.doi.org/10.1085/jgp.201311000.
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RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A.
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Stevens, Adam, Taqua Khashkhusha, Megan Sharps, Terence Garner, Peter T. Ruane i John D. Aplin. "The Human Early Maternal–Embryonic Interactome". Reproductive Medicine 4, nr 1 (16.02.2023): 40–56. http://dx.doi.org/10.3390/reprodmed4010006.
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Background: Single cell transcriptomics offers an avenue for predicting, with improved accuracy, the gene networks that are involved in the establishment of the first direct cell–cell interactions between the blastocyst and the maternal luminal epithelium. We hypothesised that in silico modelling of the maternal–embryonic interface may provide a causal model of these interactions, leading to the identification of genes associated with a successful initiation of implantation. Methods: Bulk and single cell RNA-sequencing of endometrial epithelium and scRNAseq of day 6 and 7 trophectoderm (TE) were used to model the initial encounter between the blastocyst and the maternal uterine lining epithelium in silico. In silico modelling of the maternal–embryonic interface was performed using hypernetwork (HN) analysis of genes mediating endometrial–TE interactions and the wider endometrial epithelial transcriptome. A hypernetwork analysis identifies genes that co-ordinate the expression of many other genes to derive a higher order interaction likely to be causally linked to the function. Potential interactions of TE with non-ciliated luminal cells, ciliated cells, and glandular cells were examined. Results: Prominent epithelial activities include secretion, endocytosis, ion transport, adhesion, and immune modulation. Three highly correlated clusters of 25, 22 and 26 TE-interacting epithelial surface genes were identified, each with distinct properties. Genes in both ciliated and non-ciliated luminal epithelial cells and glandular cells exhibit significant functional associations. Ciliated cells are predicted to bind to TE via galectin–glycan interaction. Day 6 and day 7 embryonic–epithelial interactomes are largely similar. The removal of aneuploid TE-derived mRNA invoked only subtle differences. No direct interaction with the maternal gland epithelial cell surface is predicted. These functional differences validate the in silico segregation of phenotypes. Single cell analysis of the epithelium revealed significant change with the cycle phase, but differences in the cell phenotype between individual donors were also present. Conclusions: A hypernetwork analysis can identify epithelial gene clusters that show correlated change during the menstrual cycle and can be interfaced with TE genes to predict pathways and processes occurring during the initiation of embryo–epithelial interaction in the mid-secretory phase. The data are on a scale that is realistic for functional dissection using current ex vivo human implantation models. A focus on luminal epithelial cells may allow a resolution to the current bottleneck of endometrial receptivity testing based on tissue lysates, which is confounded by noise from multiple diverse cell populations.
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Biedermann, Johann, Sebastian Braunbeck, Andrew J. R. Plested i Han Sun. "Nonselective cation permeation in an AMPA-type glutamate receptor". Proceedings of the National Academy of Sciences 118, nr 8 (18.02.2021): e2012843118. http://dx.doi.org/10.1073/pnas.2012843118.
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Fast excitatory synaptic transmission in the central nervous system relies on the AMPA-type glutamate receptor (AMPAR). This receptor incorporates a nonselective cation channel, which is opened by the binding of glutamate. Although the open pore structure has recently became available from cryo-electron microscopy (Cryo-EM), the molecular mechanisms governing cation permeability in AMPA receptors are not understood. Here, we combined microsecond molecular dynamic (MD) simulations on a putative open-state structure of GluA2 with electrophysiology on cloned channels to elucidate ion permeation mechanisms. Na+, K+, and Cs+ permeated at physiological rates, consistent with a structure that represents a true open state. A single major ion binding site for Na+ and K+ in the pore represents the simplest selectivity filter (SF) structure for any tetrameric cation channel of known structure. The minimal SF comprised only Q586 and Q587, and other residues on the cytoplasmic side formed a water-filled cavity with a cone shape that lacked major interactions with ions. We observed that Cl− readily enters the upper pore, explaining anion permeation in the RNA-edited (Q586R) form of GluA2. A permissive architecture of the SF accommodated different alkali metals in distinct solvation states to allow rapid, nonselective cation permeation and copermeation by water. Simulations suggested Cs+ uses two equally populated ion binding sites in the filter, and we confirmed with electrophysiology of GluA2 that Cs+ is slightly more permeant than Na+, consistent with serial binding sites preferentially driving selectivity.
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Li, Hui, Yaxin Yang, Qianlan Wang, Haoran Li, Wu Wang, Huan Zheng i Jianmin Tao. "Genome-Wide Identification of ATP-Binding Cassette (ABC) Transporter Provides Insight to Genes Related to Anthocyanin Transportation in New Teinturier Grape Germplasm ‘ZhongShan-HongYu’". Horticulturae 9, nr 5 (24.04.2023): 532. http://dx.doi.org/10.3390/horticulturae9050532.
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ATP-binding cassette (ABC) transporters are a large class of superfamily involved in a variety of biological processes with multiple functions, including phytohormone transport, heavy metal ion detoxification, and so on. Anthocyanin pigmentation in grapes is a commercially important feature of this superfamily. To elucidate the mechanisms of the VvABC gene at different stages in grape berries, we analyzed and characterized the ABC family in ‘ZhongShan-HongYu’ (ZS-HY) berries using RNA-seq analysis. In this study, a total of 146 VvABC genes were identified by comprehensive bioinformatics analysis, which outlined their gene structure, chromosomal location, conserved domains, phylogenetic relationships, and collinearity analysis. The VvABC family could be divided into eight subfamilies based on the phylogenetic analysis. Fifty-eight VvABC genes were identified from the RNA-seq data, of which 31 belong to the ABCG subfamily, 15 belong to the ABCC subfamily, 8 belong to the ABCB subfamily, 2 belong to the ABCF subfamily, and only 1 belongs to each of the ABCA and ABCD subfamilies. We used qRT-PCR to detect the expression of VvABC genes in different organs and found that it changed significantly in different organs. Phylogenetic analysis showed that genes involved in anthocyanin transport in other species were closely related to members of the ABCC subfamily. Subsequently, analysis of the promoter elements and the protein interactions of the VvABCC genes using RNA-seq was performed. This study has improved our understanding of the functions of the ABC gene family and provided a basis for the role of ABC genes in grape anthocyanin transport.
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Banerjee, Manidipa, Jeffrey A. Speir, Maggie H. Kwan, Rick Huang, Peyman P. Aryanpur, Brian Bothner i John E. Johnson. "Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate". Journal of Virology 84, nr 9 (17.02.2010): 4737–46. http://dx.doi.org/10.1128/jvi.02670-09.
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ABSTRACT Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family Nodaviridae, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membrane-disrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.
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Kaneko, Takane, Kiyotaka Toshimori i Hiroshi Iida. "Subcellular localization of MS4A13 isoform 2 in mouse spermatozoa". Reproduction 154, nr 6 (grudzień 2017): 843–57. http://dx.doi.org/10.1530/rep-17-0477.
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To identify upregulated genes during the development of spermatozoa, we performed PCR-selected subtraction analysis of testes RNA samples from 10-day-old and 12-week-old shrews. A transcript, highly homologous to two mouse transcripts, Ms4a13-1 and Ms4a13-2, was differentially regulated. Ms4a13-2, but not Ms4a13-1, was shown to be primarily expressed in mouse testes in an age-dependent manner. Ms4a13-2 cDNA contains an open-reading frame of 522 nucleotides, encoding a protein of 174 amino acids, with predicted molecular mass, 19,345 Da. MS4A13-2 protein was expressed along the periphery of nuclei of round and elongated spermatids (steps 3–16) in adult mouse testes, and in the equatorial region of the heads of fresh mature mouse spermatozoa. In addition, MS4A13-2 was found to localize to the outer acrosomal membrane in the equatorial region of heads in fresh spermatozoa. In acrosome-reacted spermatozoa, the MS4A13-2 expression extended to the entire sperm head including the postacrosomal region and acrosomal cap. MS4A family proteins are known to facilitate intracellular protein–protein interactions as ion channel/adaptor proteins by oligomerization, and have important regulatory roles in cellular growth, survival and activation. We report that the MS4A family member, MS4A13-2, may form oligomers in sperm membranes, which may be involved in an interaction with the zona pellucida or cumulus during fertilization.
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Studzińska, Sylwia, Ewelina Zawadzka, Szymon Bocian i Michał Szumski. "Synthesis and application of stationary phase for DNA-affinity chromatographic analysis of unmodified and antisense oligonucleotide". Analytical and Bioanalytical Chemistry 413, nr 20 (24.06.2021): 5109–19. http://dx.doi.org/10.1007/s00216-021-03473-7.
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AbstractThe goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses. The oligonucleotide-based stationary phase was applied for the retention studies. Unmodified oligonucleotides of different complementarity to the molecule attached as a stationary phase, as well as antisense oligonucleotides, were tested. The comparative study upon complex optimization of oligonucleotide analysis in different liquid chromatography modes was performed. Results have shown that this stationary phase may be applied for oligonucleotide analysis in hydrophilic interaction liquid chromatography and ion exchange chromatography, but no unique sequence-based selectivity was obtained. Contrary results were observed for affinity chromatography, which allowed for specific separation of the complementary strands based on hydrogen bonding and stacking interactions, where the temperature was the main factor influencing the selectivity of the separation. Furthermore, the oligonucleotide-based stationary phase may be applied for comparative antisense oligonucleotide hybridization studies to a specific RNA sequence. All of the results have shown that affinity chromatography with oligonucleotide-based stationary phases is a powerful technique for the specific base recognition of polynucleotides. Graphical abstract
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Viard, Julia, Yann Loe-Mie, Rachel Daudin, Malik Khelfaoui, Christine Plancon, Anne Boland, Francisco Tejedor i in. "Chr21 protein–protein interactions: enrichment in proteins involved in intellectual disability, autism, and late-onset Alzheimer’s disease". Life Science Alliance 5, nr 12 (1.08.2022): e202101205. http://dx.doi.org/10.26508/lsa.202101205.
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Down syndrome (DS) is caused by human chromosome 21 (HSA21) trisomy. It is characterized by a poorly understood intellectual disability (ID). We studied two mouse models of DS, one with an extra copy of the Dyrk1A gene (189N3) and the other with an extra copy of the mouse Chr16 syntenic region (Dp(16)1Yey). RNA-seq analysis of the transcripts deregulated in the embryonic hippocampus revealed an enrichment in genes associated with chromatin for the 189N3 model, and synapses for the Dp(16)1Yey model. A large-scale yeast two-hybrid screen (82 different screens, including 72 HSA21 baits and 10 rebounds) of a human brain library containing at least 107 independent fragments identified 1,949 novel protein–protein interactions. The direct interactors of HSA21 baits and rebounds were significantly enriched in ID-related genes (P-value < 2.29 × 10−8). Proximity ligation assays showed that some of the proteins encoded by HSA21 were located at the dendritic spine postsynaptic density, in a protein network at the dendritic spine postsynapse. We located HSA21 DYRK1A and DSCAM, mutations of which increase the risk of autism spectrum disorder (ASD) 20-fold, in this postsynaptic network. We found that an intracellular domain of DSCAM bound either DLGs, which are multimeric scaffolds comprising receptors, ion channels and associated signaling proteins, or DYRK1A. The DYRK1A-DSCAM interaction domain is conserved in Drosophila and humans. The postsynaptic network was found to be enriched in proteins associated with ARC-related synaptic plasticity, ASD, and late-onset Alzheimer’s disease. These results highlight links between DS and brain diseases with a complex genetic basis.
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Bahrami, Azadeh, Fatma Doğan, Deanpen Japrung i Tim Albrecht. "Solid-state nanopores for biosensing with submolecular resolution". Biochemical Society Transactions 40, nr 4 (20.07.2012): 624–28. http://dx.doi.org/10.1042/bst20120121.
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Biological cell membranes contain various types of ion channels and transmembrane pores in the 1–100 nm range, which are vital for cellular function. Individual channels can be probed electrically, as demonstrated by Neher and Sakmann in 1976 using the patch-clamp technique [Neher and Sakmann (1976) Nature 260, 799–802]. Since the 1990s, this work has inspired the use of protein or solid-state nanopores as inexpensive and ultrafast sensors for the detection of biomolecules, including DNA, RNA and proteins, but with particular focus on DNA sequencing. Solid-state nanopores in particular have the advantage that the pore size can be tailored to the analyte in question and that they can be modified using semi-conductor processing technology. This establishes solid-state nanopores as a new class of single-molecule biosensor devices, in some cases with submolecular resolution. In the present review, we discuss a few of the most important recent developments in this field and how they might be applied to studying protein–protein and protein–DNA interactions or in the context of ultra-fast DNA sequencing.