Human stimulator of interferon genes (hSTING) is a signaling adaptor protein that triggers innate immune system by response to cytosolic DNA and second messenger cyclic dinucleotides (CDNs). Natural CDNs contain purine nucleobase with different phosphodiester linkage types (3'-3', 2'-2' or mixed 2'-3'-linkages) and exhibit different binding affinity towards hSTING, ranging from micromolar to nanomolar. High-affinity CDNs are considered as suitable candidates for treatment of chronic hepatitis B and cancer. We have used molecular dynamics simulations to investigate dynamical aspects of binding of natural CDNs (specifically, 2'-2'-cGAMP, 2'-3'-cGAMP, 3'-3'-cGAMP, 3'-3'-c-di-AMP, and 3'-3'-c-di-GMP) with hSTINGwt protein. Our results revealed that CDN/hSTINGwt interactions are controlled by the balance between fluctuations (conformational changes) in the CDN ligand and the protein dynamics. Binding of different CDNs induces different degrees of conformational/dynamics changes in hSTINGwt ligand binding cavity, especially in α1-helices, the so-called lid region and α2-tails. The ligand residence time in hSTINGwt protein pocket depends on different contribution of R232 and R238 residues interacting with oxygen atoms of phosphodiester groups in ligand, water distribution around interacting charged centers (in protein residues and ligand) and structural stability of closed conformation state of hSTINGwt protein. These findings may perhaps guide design of new compounds modulating hSTING activity.Communicated by Ramaswamy H. Sarma.
- MeSH
- Dinucleoside Phosphates * chemistry MeSH
- DNA MeSH
- Humans MeSH
- Ligands MeSH
- Oligonucleotides MeSH
- Molecular Dynamics Simulation * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.
- MeSH
- Chromatography, Liquid MeSH
- COVID-19 virology MeSH
- Mass Spectrometry MeSH
- Humans MeSH
- Methyltransferases genetics metabolism MeSH
- Methylation MeSH
- Gene Expression Regulation, Viral MeSH
- RNA Caps MeSH
- RNA, Viral genetics MeSH
- SARS-CoV-2 enzymology genetics MeSH
- Substrate Specificity MeSH
- Viral Nonstructural Proteins genetics metabolism MeSH
- Viral Regulatory and Accessory Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.
- MeSH
- Adenine chemistry metabolism MeSH
- Aptamers, Nucleotide chemical synthesis genetics MeSH
- Cytosine chemistry metabolism MeSH
- Deoxyribonucleosides chemistry genetics metabolism MeSH
- Dinucleoside Phosphates chemistry genetics metabolism MeSH
- DNA-Directed DNA Polymerase genetics metabolism MeSH
- DNA chemistry genetics metabolism MeSH
- Guanine chemistry metabolism MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Base Pairing MeSH
- Polymerase Chain Reaction MeSH
- Polymers chemical synthesis metabolism MeSH
- DNA Replication * MeSH
- Base Sequence MeSH
- Uracil chemistry metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- RNA, Bacterial MeSH
- Biochemistry MeSH
- Dinucleoside Phosphates MeSH
- Escherichia coli MeSH
- Research MeSH
- Publication type
- Popular Work MeSH
- Interview MeSH
Dinucleoside polyphosphates (NpnNs) were discovered 50 years ago in all cells. They are often called alarmones, even though the molecular target of the alarm has not yet been identified. Recently, we showed that they serve as noncanonical initiating nucleotides (NCINs) and fulfill the role of 5' RNA caps in Escherichia coli. Here, we present molecular insight into their ability to be used as NCINs by T7 RNA polymerase in the initiation phase of transcription. In general, we observed NpnNs to be equally good substrates as canonical nucleotides for T7 RNA polymerase. Surprisingly, the incorporation of ApnGs boosts the production of RNA 10-fold. This behavior is due to the pairing ability of both purine moieties with the -1 and +1 positions of the antisense DNA strand. Molecular dynamic simulations revealed noncanonical pairing of adenosine with the thymine of the DNA.
- MeSH
- Bacteriophage T7 enzymology MeSH
- Dinucleoside Phosphates genetics metabolism MeSH
- DNA-Directed RNA Polymerases genetics metabolism MeSH
- DNA metabolism MeSH
- Transcription Initiation, Genetic * MeSH
- Base Pairing MeSH
- RNA Caps genetics MeSH
- RNA genetics metabolism MeSH
- Molecular Dynamics Simulation MeSH
- Protein Binding MeSH
- Viral Proteins genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.
- MeSH
- RNA, Bacterial genetics MeSH
- Dinucleoside Phosphates genetics MeSH
- DNA-Directed RNA Polymerases genetics MeSH
- Escherichia coli genetics MeSH
- Acid Anhydride Hydrolases metabolism MeSH
- Nucleic Acid Conformation MeSH
- Methylation MeSH
- Escherichia coli Proteins metabolism MeSH
- RNA Caps genetics MeSH
- RNA Stability MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Epilepsy is a multifactorial disorder associated with neuronal hyperexcitability that affects more than 1% of the human population. It has long been known that adenosine can reduce seizure generation in animal models of epilepsies. However, in addition to various side effects, the instability of adenosine has precluded its use as an anticonvulsant treatment. Here we report that a stable analogue of diadenosine-tetraphosphate: AppCH2ppA effectively suppresses spontaneous epileptiform activity in vitro and in vivo in a Tuberous Sclerosis Complex (TSC) mouse model (Tsc1+/-), and in postsurgery cortical samples from TSC human patients. These effects are mediated by enhanced adenosine signaling in the cortex post local neuronal adenosine release. The released adenosine induces A1 receptor-dependent activation of potassium channels thereby reducing neuronal excitability, temporal summation, and hypersynchronicity. AppCH2ppA does not cause any disturbances of the main vital autonomous functions of Tsc1+/- mice in vivo. Therefore, we propose this compound to be a potent new candidate for adenosine-related treatment strategies to suppress intractable epilepsies.
- MeSH
- Adenosine physiology MeSH
- Anticonvulsants administration & dosage MeSH
- Dinucleoside Phosphates administration & dosage MeSH
- Potassium Channels physiology MeSH
- Tuberous Sclerosis Complex 1 Protein genetics MeSH
- Humans MeSH
- Membrane Potentials drug effects MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Neocortex drug effects physiopathology MeSH
- Neurons drug effects physiology MeSH
- Receptor, Adenosine A1 physiology MeSH
- Signal Transduction drug effects MeSH
- Seizures physiopathology prevention & control MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Density functional theory (DFT) has been applied to study the conformational dependence of 31P chemical shift tensors in B-DNA. The gg and gt conformations of backbone phosphate groups representing BI- and BII-DNA have been examined. Calculations have been carried out on static models of dimethyl phosphate (dmp) and dinucleoside-3',5'-monophosphate with bases replaced by hydrogen atoms in vacuo as well as in an explicit solvent. Trends in 31P chemical shift anisotropy (CSA) tensors with respect to the backbone torsion angles alpha, zeta, beta, and epsilon are presented. Although these trends do not change qualitatively upon solvation, quantitative changes result in the reduction of the chemical shift anisotropy. For alpha and zeta in the range from 270 degrees to 330 degrees and from 240 degrees to 300 degrees , respectively, the delta22 and delta33 principal components vary within as much as 30 ppm, showing a marked dependence on backbone conformation. The calculated 31P chemical shift tensor principal axes deviate from the axes of O-P-O bond angles by at most 5 degrees . For solvent models, our results are in a good agreement with experimental estimates of relative gg and gt isotropic chemical shifts. Solvation also brings the theoretical deltaiso of the gg conformation closer to the experimental gg data of barium diethyl phosphate.
