NMR Studies of Genomic RNA in 3' Untranslated Region Unveil Pseudoknot Structure that Initiates Viral RNA Replication in SARS-CoV-2.

In the 3' untranslated region of the SARS-CoV-2 virus RNA genome, genomic RNA replication is initiated in the highly conserved region called 3'PK, containing three stem structures (P1pk, P2, and P5). According to one proposed mechanism, P1pk and distal P2 stems switch their structure to a pseudoknot through base-pairing, thereby initiating transcription by recruiting RNA-dependent RNA polymerase complexed with nonstructural proteins (nsp)7 and nsp8. However, experimental evidence of pseudoknot formation or structural switching is unavailable.

An NMR-based approach reveals the core structure of the functional domain of SINEUP lncRNAs.

Long non-coding RNAs (lncRNAs) are attracting widespread attention for their emerging regulatory, transcriptional, epigenetic, structural and various other functions. Comprehensive transcriptome analysis has revealed that retrotransposon elements (REs) are transcribed and enriched in lncRNA sequences. However, the functions of lncRNAs and the molecular roles of the embedded REs are largely unknown.

Structures and Catalytic Activities of Complexes between Heme and All Parallel-Stranded Monomeric G-Quadruplex DNAs.

Heme in its ferrous and ferric states [heme(Fe) and heme(Fe), respectively] binds selectively to the 3'-terminal G-quartet of all parallel-stranded monomeric G-quadruplex DNAs formed from inosine(I)-containing sequences, i.e., d(TAGGGTGGGTTGGGTGIG) DNA(18mer) and d(TAGGGTGGGTTGGGTGIGA) DNA(18mer/A), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet, to form 1:1 complexes [heme-DNA(18mer) and heme-DNA(18mer/A) complexes, respectively].

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation.

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA.

High Glucose Stimulates Mineralocorticoid Receptor Transcriptional Activity Through the Protein Kinase C β Signaling.

Activation of mineralocorticoid receptor (MR) is shown in resistant hypertension including diabetes mellitus. Although protein kinase C (PKC) signaling is involved in the pathogenesis of diabetic complications, an association between PKC and MR is not known. Activation of PKCα and PKCβ by TPA (12-O-Tetradecanoylphorbol 13-acetate) increased MR proteins and its transcriptional activities in HEK293-MR cells.

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions.

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell 'stemness' and tumorigenesis. Msi1 reportedly binds to the 3'-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex.

Prediction of recovery time from hypoglycemia in patients with insulin overdose.

Insulin overdose results in prolonged hypoglycemia. We hypothesized that if a huge amount of insulin is subcutaneously injected, the duration of hypoglycemia depends on the dose of insulin rather than the type of insulin. We conducted a literature review of insulin overdose and 33 cases were included in this study.

Structural analysis of Musashi-RNA complex on the basis of long-range structural information.

Musashi protein is supposed to be involved in the regulation of differentiation of neural stem cells. Musashi binds to 3' untranslated region of target mRNA and represses the translation of mRNA. Musashi has two tandem RNA-binding domains (RBDs), RBD1 and RBD2.

Unexpected A-form formation of 4'-thioDNA in solution, revealed by NMR, and the implications as to the mechanism of nuclease resistance.

Fully modified 4'-thioDNA, an oligonucleotide only comprising 2'-deoxy-4'-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4'-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4'-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available.

Structure of 4'-thioDNA which exhibits endonuclease resistance.

4'-thio DNA consisting of 2'-deoxy-4'-thionucleosides exhibits resistance to both endonuclease and 3'-exonuclease cleavages. Interestingly, we found that 4'-thioDNA duplex behaved like RNA molecules in hybridization properties and structural aspects. Here, we have determined the structure of 4'-thioDNA duplex in solution by NMR.

Interactions with RNA/DNA of proteins involved in the regulation of transcription, translation and telomere elongation.

Interactions with DNA and RNA of three different proteins involved in the regulation of (1) transcription, (2) translation, and (3) telomere elongation were examined by NMR. In the first case, the combination of structural determination, dynamical analysis on the basis of relaxation data and identification of interactive surface for wild and phosphorylation-mimicking mutant proteins has given the insight on the increase of DNA-binding affinity through phosphorylation of the protein. In the second case, the arrangement of two tandem domains interacting with RNA has been determined with residual dipolar couplings and paramagnetic relaxation enhancement, which has given the idea on how the two tandem domains recognize the target RNA.

Structural analysis of DNA containing (6-4) photoproduct in complex with distamycin A by NMR.

We reported that distamycin A can bind to DNA containing the (6-4) photoproduct, one of the major UV lesions in DNA, by means of CD spectroscopy. Here, we have analyzed the structure of DNA containing the (6-4) photoproduct in complex with distamycin A by NMR. Broadening of NMR resonances and large chemical shift perturbation have been observed for residues located close to the (6-4) photoproduct in DNA, indicating the binding to this region.

Dynamics and thermodynamics of dimerization of parallel G-quadruplexed DNA through pi-pi stacking interaction.

DNA fragments of d(TTAG(n)) (n = 3-5) form all-parallel G-quadruplexed structure in the presence of K+. We found that G-quadruplexed d(TTAG(n)) forms dimer through end-to-end stacking of the 3'-terminal G-tetrads. In this study, we report the structure of the dimer, and the dynamics and thermodynamics of the dimerization.

Structural and functional characterization of novel G-quadruplexed DNA-heme coordination complex.

We analyzed the coordination structure of G-quadruplexed DNA-heme complex which exhibits remarkably similar spectroscopic characters to hemoprotein such as myoglobin. We found that some exogenous ligands can be accommodated at the 6th coordination site of heme in the G-quadruplexed DNA-heme complex with a DNA base coordinated to the 5th site. The G-quadruplexed DNA-hemin complex is expected to play a role as a novel DNAzyme.

Study on the complexation between DNA and cationic porphyrin derivatives.

A water-soluble cationic porphyrin, 5,10,15,20-Tetra(N-methylpyridinium-4-yl)-21H,23H-porphyrin has been shown to intercalate selectively into the A3-G4 gap of C-quadruplexed DNA d(TTAGGG)4.

Formation of a complex of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin with G-quadruplex DNA.

A water-soluble cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TmPyP4), has been studied extensively because of its unique physicochemical properties that lead to interactions with nucleic acids, as well as its therapeutic application. Formation of a complex between TmPyP4 and parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized in an effort to elucidate the mode of molecular recognition between TmPyP4 and the DNA. The study demonstrated that TmPyP4 intercalates into the A3pG4 step of [d(TTAGGG)]4 with an association constant of 6.

Dynamics and thermodynamics of dimerization of parallel G-quadruplexed DNA formed from d(TTAGn) (n=3-5).

Parallel G-quadruplexes formed from oligonucleotide sequences, d(TTAGn), where n = 3-5, have been shown to form a dimer through end-to-end stacking of 3'-terminal G-tetrads. The monomers and dimers of the G-quadruplexes are in dynamic equilibrium with an exchange rate of approximately 1 s-1. A thermodynamic study demonstrated that the dimerization of the G-quadruplexes is largely enthalpic in origin.

Binding of 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin to an AT-rich region of a duplex DNA.

Characterization of the interaction between DNA and small organic compounds is of considerable importance for gaining insights into the mechanism underlying molecular recognition, which could be highly relevant to drug design. In the present study, the interaction of a water-soluble cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin, with a self-complementary duplex DNA, d(GCTTAAGC)2, has been investigated by means of absorption, circular dichroism, and NMR spectroscopies. The optical studies indicated that TMPyP binds to the TTAA region of d(GCTTAAGC)2 with a binding constant of 2.

Study on interaction of a cationic porphyrin with DNA.

5,10,15,20-Tetra(N-methyl-pyridinium-4-yl)-21H,23H-porphyrin has shown to bind to the major groove of AT-rich DNA predominantly through electrostatic interaction between positively charged N-methyl pyridinium moieties of the porphyrin and negatively charged phosphodiester backbone. Solution structure of the complex between the porphyrin and a double-stranded DNA fragment has been inferred from the measurements of the mixing time-dependent intermolecular nuclear Overhauser effects (NOEs).

Coordination complex between haemin and parallel-quadruplexed d(TTAGGG).

Haemin, iron(III)-protoporphyrin IX complex, and parallel-quadruplexed d(TTAGGG) have been shown to form a stable coordination complex which exhibits spectroscopic properties remarkably similar to those of haemoproteins.