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
- dinukleosidfosfáty * chemie MeSH
- DNA MeSH
- lidé MeSH
- ligandy MeSH
- oligonukleotidy MeSH
- simulace molekulární dynamiky * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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
- adenin chemie metabolismus MeSH
- aptamery nukleotidové chemická syntéza genetika MeSH
- cytosin chemie metabolismus MeSH
- deoxyribonukleosidy chemie genetika metabolismus MeSH
- dinukleosidfosfáty chemie genetika metabolismus MeSH
- DNA-dependentní DNA-polymerasy genetika metabolismus MeSH
- DNA chemie genetika metabolismus MeSH
- guanin chemie metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- párování bází MeSH
- polymerázová řetězová reakce MeSH
- polymery chemická syntéza metabolismus MeSH
- replikace DNA * MeSH
- sekvence nukleotidů MeSH
- uracil chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- bakteriální RNA MeSH
- biochemie MeSH
- dinukleosidfosfáty MeSH
- Escherichia coli MeSH
- výzkum MeSH
- Publikační typ
- populární práce MeSH
- rozhovory 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
- bakteriofág T7 enzymologie MeSH
- dinukleosidfosfáty genetika metabolismus MeSH
- DNA řízené RNA-polymerasy genetika metabolismus MeSH
- DNA metabolismus MeSH
- iniciace genetické transkripce * MeSH
- párování bází MeSH
- RNA čepičky genetika MeSH
- RNA genetika metabolismus MeSH
- simulace molekulární dynamiky MeSH
- vazba proteinů MeSH
- virové proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- bakteriální RNA genetika MeSH
- dinukleosidfosfáty genetika MeSH
- DNA řízené RNA-polymerasy genetika MeSH
- Escherichia coli genetika MeSH
- hydrolasy působící na anhydridy kyselin metabolismus MeSH
- konformace nukleové kyseliny MeSH
- metylace MeSH
- proteiny z Escherichia coli metabolismus MeSH
- RNA čepičky genetika MeSH
- stabilita RNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- adenosin fyziologie MeSH
- antikonvulziva aplikace a dávkování MeSH
- dinukleosidfosfáty aplikace a dávkování MeSH
- draslíkové kanály fyziologie MeSH
- hamartin genetika MeSH
- lidé MeSH
- membránové potenciály účinky léků MeSH
- myši transgenní MeSH
- myši MeSH
- neokortex účinky léků patofyziologie MeSH
- neurony účinky léků fyziologie MeSH
- receptor adenosinový A1 fyziologie MeSH
- signální transdukce účinky léků MeSH
- záchvaty patofyziologie prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
