Search

The application of oncolytic peptides has become a powerful approach to induce complete and long-lasting remission in multiple types of carcinomas, as affirmed by the appearance of tumor-associated antigens and adenosine triphosphate (ATP) in large quantities, which jumpstarts the cancer-immunity cycle. However, the ATP breakdown product adenosine is a significant contributor to forming the immunosuppressive tumor microenvironment, which substantially weakens peptide-driven oncolytic immunotherapy. In this study, a lipid-coated micelle (CA@TLM) loaded with a stapled oncolytic peptide (PalAno) and an adenosine 2A receptor (A2AR) inhibitor (CPI-444) is devised to enact tumor-targeted oncolytic immunotherapy and to overcome adenosine-mediated immune suppression simultaneously. The CA@TLM micelle accumulates in tumors with high efficiency, and the acidic tumor microenvironment prompts the rapid release of PalAno and CPI-444. Subsequently, PalAno induces swift membrane lysis of tumor cells and the release of antigenic materials. Meanwhile, CPI-444 blocks the activation of the immunosuppressive adenosine-A2AR signaling pathway. This combined approach exhibits pronounced synergy at stalling tumor growth and metastasis in animal models for triple-negative breast cancer and melanoma, providing a novel strategy for enhanced oncolytic immunotherapy., (© 2024 Wiley‐VCH GmbH.)

Immunogenic cell death (ICD) often results in the production and accumulation of adenosine (ADO), a byproduct that negatively impacts the therapeutic effect as well as facilitates tumor development and metastasis. Here, an innovative strategy is elaborately developed to effectively activate ICD while avoiding the generation of immunosuppressive adenosine. Specifically, ZIF-90, an ATP-responsive consumer, is synthesized as the core carrier to encapsulate AB680 (CD73 inhibitor) and then coated with an iron-polyphenol layer to prepare the ICD inducer (AZTF), which is further grafted onto prebiotic bacteria via the esterification reaction to obtain the engineered biohybrid (Bc@AZTF). Particularly, the designed Bc@AZTF can actively enrich in tumor sites and respond to the acidic tumor microenvironment to offload AZTF nanoparticles, which can consume intracellular ATP (iATP) content and simultaneously inhibit the ATP-adenosine axis to reduce the accumulation of adenosine, thereby alleviating adenosine-mediated immunosuppression and strikingly amplifying ICD effect. Importantly, the synergy of anti-PD-1 (αPD-1) with Bc@AZTF not only establishes a collaborative antitumor immune network to potentiate effective tumoricidal immunity but also activates long-lasting immune memory effects to manage tumor recurrence and rechallenge, presenting a new paradigm for ICD treatment combined with adenosine metabolism., (© 2024 Wiley‐VCH GmbH.)

The SARS-CoV-2 genome has been shown to be m 6 A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m 6 A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The continuous deciphering of crucial biological roles of RNA modifications and their involvement in various pathological conditions, together with their key roles in the use of RNA-based therapeutics, has reignited interest in studying the occurrence and identity of non-canonical ribonucleoside structures during the past years. Discovery and structural elucidation of new modified structures is usually achieved by combination of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) at the nucleoside level and stable isotope labeling experiments. This approach, however, has its pitfalls as demonstrated in the course of the present study: we structurally elucidated a new nucleoside structure that showed significant similarities to the family of (c)t 6 A modifications and was initially considered a genuine modification, but subsequently turned out to be an in vitro formed glycerol ester of t 6 A. This artifact is generated from ct 6 A during RNA hydrolysis upon addition of enzymes stored in glycerol containing buffers in a mildly alkaline milieu, and was moreover shown to undergo an intramolecular transesterification reaction. Our results demand for extra caution, not only in the discovery of new RNA modifications, but also with regard to the quantification of known modified structures, in particular chemically labile modifications, such as ct 6 A, that might suffer from exposure to putatively harmless reagents during the diverse steps of sample preparation., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

N1-methyladenosine (m 1 A) modification is one of the most prevalent epigenetic modifications on RNA. Given the vital role of m 1 A modification in RNA processing such as splicing, stability and translation, developing a precise and controllable m 1 A editing tool is pivotal for in-depth investigating the biological functions of m 1 A. In this study, we developed an abscisic acid (ABA)-inducible and reversible m 1 A demethylation tool (termed AI-dm 1 A), which targets specific transcripts by combining the chemical proximity-induction techniques with the CRISPR/dCas13b system and ALKBH3. We successfully employed AI-dm 1 A to selectively demethylate the m 1 A modifications at A8422 of MALAT1 RNA, and this demethylation process could be reversed by removing ABA. Furthermore, we validated its demethylation function on various types of cellular RNAs including mRNA, rRNA and lncRNA. Additionally, we used AI-dm 1 A to specifically demethylate m 1 A on ATP5D mRNA, which promoted ATP5D expression and enhanced the glycolysis activity of tumor cells. Conversely, by replacing the demethylase ALKBH3 with methyltransferase TRMT61A, we also developed a controllable m 1 A methylation tool, namely AI-m 1 A. Finally, we caged ABA by 4,5-dimethoxy-2-nitrobenzyl (DMNB) to achieve light-inducible m 1 A methylation or demethylation on specific transcripts. Collectively, our m 1 A editing tool enables us to flexibly study how m 1 A modifications on specific transcript influence biological functions and phenotypes., (© 2024 Wiley-VCH GmbH.)

METTL3, a primary methyltransferase catalyzing the RNA N6-methyladenosine (m6A) modification, has been identified as an oncogene in several cancer types and thus nominated as a potentially effective target for therapeutic inhibition. However, current options using this strategy are limited. In this study, we targeted protein-protein interactions at the METTL3-METTL14 binding interface to inhibit complex formation and subsequent catalysis of the RNA m6A modification. Among candidate peptides, RM3 exhibited the highest anti-cancer potency, inhibiting METTL3 activity while also facilitating its proteasomal degradation. We then designed a stapled peptide inhibitor (RSM3) with enhanced peptide stability and formation of the α-helical secondary structure required for METTL3 interaction. Functional and transcriptomic analysis in vivo indicated that RSM3 induced upregulation of programmed cell death-related genes while inhibiting cancer-promoting signals. Furthermore, tumor growth was significantly suppressed while apoptosis was enhanced upon RSM3 treatment, accompanied by increased METTL3 degradation, and reduced global RNA methylation levels in two in vivo tumor models. This peptide inhibitor thus exploits a mechanism distinct from other small-molecule competitive inhibitors to inhibit oncogenic METTL3 activity. Our findings collectively highlight the potential of targeting METTL3 in cancer therapies through peptide-based inhibition of complex formation and proteolytic degradation., (© 2024 Wiley-VCH GmbH.)

N6-methyladenosine (m 6 A) on RNAs plays an important role in regulating various biological processes and CRIPSR technology has been employed for programmable m 6 A editing. However, the bulky size of CRISPR protein and constitutively expressed CRISPR/RNA editing enzymes can interfere with the native function of target RNAs and cells. Herein, we reported a conditional m 6 A editing platform (FKBP*-dCas13b-ALK) based on a ligand stabilized dCas13 editor. The inducible expression of this m 6 A editing system was achieved by adding or removing the Shield-1 molecule. We further demonstrated that the targeted recruitment of dCas13b-m 6 A eraser fusion protein and site-specific m 6 A erasing were achieved under the control of Shield-1. Moreover, the release and degradation of dCas13b fusion protein occurred faster than the restoration of m 6 A on the target RNAs after Shield-1 removal, which provides an ideal opportunity to study the m 6 A function with minimal steric interference from bulky dCas13b fusion protein., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Traditionally, molecular recognition between the orthosteric site of adenosine receptors and their endogenous ligand occurs with a 1 : 1 stoichiometry. Inspired by previous mechanistic insights derived from supervised molecular dynamics (SuMD) simulations, which suggested an alternative 2 : 1 binding stoichiometry, we synthesized BRA1, a bis-ribosyl adenosine derivative, tested its ability to bind to and activate members of the adenosine receptor family, and rationalized its activity through molecular modeling., (© 2023 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

The fields of RNA modification and RNA damage both exhibit a plethora of non-canonical nucleoside structures. While RNA modifications have evolved to improve RNA function, the term RNA damage implies detrimental effects. Based on stable isotope labelling and mass spectrometry, we report the identification and characterisation of 2-methylthio-1,N6-ethenoadenosine (ms 2 ϵA), which is related to 1,N6-ethenoadenine, a lesion resulting from exposure of nucleic acids to alkylating chemicals in vivo. In contrast, a sophisticated isoprene labelling scheme revealed that ms 2 ϵA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2-methylthio-N6-isopentenyladenosine (ms 2 i 6 A). The relative abundance of ms 2 ϵA in tRNAs from translating ribosomes suggests reduced function in comparison to its parent RNA modification, establishing the nature of the new structure in a newly perceived overlap of the two previously separate fields, namely an RNA modification damage., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Gliflozins are successfully marketed antidiabetic agents with a reported neuroprotective effect, and this study tests their blood-brain barrier crossing ability. Henceforward, a computational hypothesis interpreting their effects was reasonable after failure to cross into the brain. A chromatographic bioassay for canagliflozin, dapagliflozin, and empagliflozin was developed, validated, and applied to the rat's and rat's plasma and brain. HPLC method robustness was tested over two levels using Design of Experiment on MINITAB. It is the first method for gliflozins' detection in rats' brain tissue. The method was applied on 18 rats and six for each drug. Concentrations in plasma were determined but neither of them was detected in brain at the described chromatographic conditions. A computational study for the three drugs was endorsing two techniques. First, ligand-based target fishing reveals possible targets for gliflozins. They showed an ability to bind with human equilibrative nucleoside transporter 1, a regulator of adenosine extracellularly. Second, a docking study was carried out on this protein receptor. Results showed perfect alignment with a minimum of one hydrogen bond. Dapagliflozin achieved the lowest energy score with two hocking hydrogen bonds. This is proposing gliflozins ability to regulate equilibrative nucleoside transporter 1 receptors in peripheries, elevating the centrally acting neuroprotective adenosine., (© 2023 Wiley-VCH GmbH.)

Since early 2020, scientists have strived to find an effective solution to fight SARS-CoV-2, in particular by developing reliable vaccines that inhibit the spread of the disease and repurposing drugs for combatting its effects on the human body. The antiviral prodrug Remdesivir is still the most widely used therapeutic during the early stages of the infection. However, the current synthetic routes rely on the use of protecting groups, air-sensitive reagents, and cryogenic conditions, thus impeding a cost-efficient supply to patients. We have, therefore, focused on the development of a straightforward, direct addition of (hetero)arenes to unprotected sugars. Here we report a silylium-catalyzed and completely stereoselective C-glycosylation that initially yields the open-chain polyols, which can be selectively cyclized to provide either the kinetic α-furanose or the thermodynamically favored β-anomer. The method significantly expedites the synthesis of Remdesivir precursor GS-441524 after a subsequent Mn-catalyzed C-H oxidation and deoxycyanation., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

N 6 -methyladenosine (m 6 A) modification-the most prevalent mammalian RNA internal modification-plays key regulatory roles in mRNA metabolism. Current approaches for m 6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m 6 A-specific in situ hybridization mediated proximity ligation assay (m 6 AISH-PLA) for cellular imaging of m 6 A RNA, allowing to identify m 6 A modification at specific location in RNAs and image m 6 A RNA with single-cell and single-molecule resolution. Using m 6 AISH-PLA, we investigated the m 6 A level and subcellular location of HSP70 RNA103-m 6 A in response to heat shock stress, and found an increased m 6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m 6 AISH-PLA can serve in the study of m 6 A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis., (© 2021 Wiley-VCH GmbH.)

N 1 -methyladenosine (m 1 A) is a prevalent and reversible RNA modification, which plays a crucial role in the regulation of RNA fate and gene expression. However, the lack of tools to precisely manipulate m 1 A sites in specific transcripts has hindered efforts to clarify the association between a specific m 1 A-modified transcript and its phenotypic outcomes. Here we develop a CRISPR-Cas13d-based tool called reengineered m 1 A modification valid eraser (termed "REMOVER") for targeted m 1 A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m 1 A demethylase ALKBH3, and the dCasRx-ALKBH3 fusion protein can mediate potent demethylation of m 1 A-modified RNAs. We further find that REMOVER can specifically demethylate m 1 A of MALAT1 and PRUNE1 RNAs, thereby significantly increasing their stability. Our study establishes REMOVER as a tool for targeted RNA demethylation of specific m 1 A-modified transcripts, which enables further elucidation of the relationship between m 1 A modification of specific transcripts and their phenotypic outcomes., (© 2021 Wiley-VCH GmbH.)

Adenosine-to-inosine (A-to-I) editing is a conserved eukaryotic RNA modification that contributes to development, immune response, and overall cellular function. Here, we utilize Endonuclease V (EndoV), which binds specifically to inosine in RNA, to develop an EndoV-linked immunosorbency assay (EndoVLISA) as a rapid, plate-based chemiluminescent method for measuring global A-to-I editing signatures in cellular RNA. We first optimize and validate our assay with chemically synthesized oligonucleotides. We then demonstrate rapid detection of inosine content in treated cell lines, demonstrating equivalent performance against current standard RNA-seq approaches. Lastly, we deploy our EndoVLISA for profiling differential A-to-I RNA editing signatures in normal and diseased human tissue, illustrating the utility of our platform as a diagnostic bioassay. Together, the EndoVLISA method is cost-effective, straightforward, and utilizes common laboratory equipment, offering a highly accessible new approach for studying A-to-I editing. Moreover, the multi-well plate format makes this the first assay amenable for direct high-throughput quantification of A-to-I editing for applications in disease detection and drug development., (© 2021 Wiley-VCH GmbH.)

Arsenosugars are a group of arsenic-containing ribosides that are found predominantly in marine algae but also in terrestrial organisms. It has been proposed that arsenosugar biosynthesis involves a key intermediate 5'-deoxy-5'-dimethylarsinoyl-adenosine (DDMAA), but how DDMAA is produced remains elusive. Now, we report characterization of ArsS as a DDMAA synthase, which catalyzes a radical S-adenosylmethionine (SAM)-mediated alkylation (adenosylation) of dimethylarsenite (DMAs III ) to produce DDMAA. This radical-mediated reaction is redox neutral, and multiple turnover can be achieved without external reductant. Phylogenomic and biochemical analyses revealed that DDMAA synthases are widespread in distinct bacterial phyla with similar catalytic efficiencies; these enzymes likely originated from cyanobacteria. This study reveals a key step in arsenosugar biosynthesis and also a new paradigm in radical SAM chemistry, highlighting the catalytic diversity of this superfamily of enzymes., (© 2021 Wiley-VCH GmbH.)

Binuclear lanthanide complexes of Eu(III) and Sm(III) were obtained in the presence of chiral ligand 1,2-(R,R+S,S)-N,N'-bis(2-pyridylmethylene),2-diamine. An unusual structure of the Eu(III) compound with two lanthanide atoms connected through two chlorines was determined by X-ray crystallography. In solution, the dimer coexists with a monomeric complex, and the stability of the binuclear form depends on the solvent and concentration. The dimer-monomer equilibrium was monitored by circularly polarized luminescence (CPL) measured on a Raman optical activity (ROA) spectrometer, where both forms provided large CPL anisotropic ratios of up to 5.6×10 -2 . Monomer formation was favored in water, whereas the dimer was stabilized in methanol. When mixed with adenosine phosphates, AMP gave much smaller CPL than ADP and ATP, indicating a high affinity of the Eu (III) complex for the phosphate group, which in connection with the ROA/CPL technique can be developed into a bioanalytical probe., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Approaches that enable innate repair mechanisms hold great potential for tissue repair. Herein, biomaterial-assisted sequestration of small molecules is described to localize pro-regenerative signaling at the injury site. Specifically, a synthetic biomaterial containing boronate molecules is designed to sequester adenosine, a small molecule ubiquitously present in the human body. The biomaterial-assisted sequestration of adenosine leverages the transient surge of extracellular adenosine following injury to prolong local adenosine signaling. It is demonstrated that implantation of the biomaterial patch following injury establishes an in situ stockpile of adenosine, resulting in accelerated healing by promoting both osteoblastogenesis and angiogenesis. The adenosine content within the patch recedes to the physiological level as the tissue regenerates. In addition to sequestering endogenous adenosine, the biomaterial is also able to deliver exogenous adenosine to the site of injury, offering a versatile solution to utilizing adenosine as a potential therapeutic for tissue repair., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adenosylhopane is a crucial intermediate in the biosynthesis of bacteriohopanepolyols, which are widespread prokaryotic membrane lipids. Herein, it is demonstrated that reconstituted HpnH, a putative radical S-adenosyl-l-methionine (SAM) enzyme, commonly encoded in the hopanoid biosynthetic gene cluster, converts diploptene into adenosylhopane in the presence of SAM, flavodoxin, flavodoxin reductase, and NADPH. NMR spectra of the enzymatic reaction product were identical to those of synthetic (22R)-adenosylhopane, indicating that HpnH catalyzes stereoselective C-C formation between C29 of diploptene and C5' of 5'-deoxyadenosine. Further, the HpnH reaction in D 2 O-containing buffer revealed that a D atom was incorporated at the C22 position of adenosylhopane. Based on these results, we propose a radical addition reaction mechanism catalyzed by HpnH for the formation of the C 35 bacteriohopane skeleton., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite the large variety of modified nucleosides that have been reported, the preparation of constrained 4'-spirocyclic adenosine analogues has received very little attention. We discovered that the [2+2]-cycloaddition of dichloroketene on readily available 4'-exo-methylene furanose sugars efficiently results in the diastereoselective formation of novel 4'-spirocyclobutanones. The reaction mechanism was investigated via density functional theory (DFT) and found to proceed either via a non-synchronous or stepwise reaction sequence, controlled by the stereochemistry at the 3'-position of the sugar substrate. The obtained dichlorocyclobutanones were converted into nucleoside analogues, providing access to a novel class of chiral 4'-spirocyclobutyl adenosine mimetics in eight steps from commercially available sugars. Assessment of the biological activity of designed 4'-spirocyclic adenosine analogues identified potent inhibitors for protein methyltransferase target PRMT5., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two different artificial predation strategies are spatially and temporally coupled to generate a simple tit-for-tat mechanism in a ternary protocell network capable of antagonistic enzyme-mediated interactions. The consortium initially consists of protease-sensitive glucose-oxidase-containing proteinosomes (1), non-interacting pH-sensitive polypeptide/mononucleotide coacervate droplets containing proteinase K (2), and proteinosome-adhered pH-resistant polymer/polysaccharide coacervate droplets (3). On receiving a glucose signal, secretion of protons from 1 triggers the disassembly of 2 and the released protease is transferred to 3 to initiate a delayed contact-dependent killing of the proteinosomes and cessation of glucose oxidase activity. Our results provide a step towards complex mesoscale dynamics based on programmable response-retaliation behavior in artificial protocell consortia., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The synthesis of the protected form of 2-methylthio-N 6 -threonylcarbamoyl adenosine (ms 2 t 6 A) was developed starting from adenosine or guanosine by using the optimized carbamate method and, for the first time, an isocyanate route. The hypermodified nucleoside was subsequently transformed into the protected ms 2 t 6 A-phosphoramidite monomer and used in a large-scale synthesis of the precursor 17nt ms 2 t 6 A-oligonucleotide (the anticodon stem and loop fragment of tRNA Lys from T. brucei). Finally, stereochemically secure ms 2 t 6 A→ms 2 ct 6 A cyclization at the oligonucleotide level efficiently afforded a tRNA fragment bearing the ms 2 ct 6 A unit. The applied post-synthetic approach provides two sequentially homologous ms 2 t 6 A- and ms 2 ct 6 A-oligonucleotides that are suitable for further comparative structure-activity relationship studies., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Iron is essential for the pathogenicity and virulence of Mycobacterium tuberculosis, which synthesises salicyl-capped siderophores (mycobactins) to acquire this element from the host. MbtA is the adenylating enzyme that catalyses the initial reaction of mycobactin biosynthesis and is solely expressed by mycobacteria. A 3200-member library comprised of lead-like, structurally diverse compounds was screened against M. tuberculosis for whole-cell inhibitory activity. A set of 846 compounds that inhibited the tubercle bacilli growth were then tested for their ability to bind to MbtA using a fluorescence-based thermal shift assay and NMR-based Water-LOGSY and saturation transfer difference (STD) experiments. We identified an attractive hit molecule, 5-hydroxyindol-3-ethylamino-(2-nitro-4-trifluoromethyl)benzene (5), that bound with high affinity to MbtA and produced a MIC 90 value of 13 μm. The ligand was docked into the MbtA crystal structure and displayed an excellent fit within the MbtA active pocket, adopting a binding mode different from that of the established MbtA inhibitor Sal-AMS., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Poly(propyleneimine) glycodendrimers are proposed as nanocarriers for triphosphate forms of anticancer adenosine analogues to improve the efficiency of chemotherapy and to overcome drug resistance mechanisms. This approach has proven successful for fludarabine administration-an autonomous way of cellular entry of a nucleotide-dendrimer noncovalent complex enables an increase in the intracellular accumulation and cytotoxic activity of the active metabolite of the drug. However, the attempt to apply an analogous strategy for clofarabine results in the inhibition of drug activity. To better understand this phenomenon, characterization and comparison of drug-dendrimer complexes were needed to indicate the differences in their surface properties and the strengths of fludarabine-dendrimer and clofarabine-dendrimer interactions. Here, zeta potential measurements, ultrafiltration, and asymmetric flow field-flow fractionation are applied to determine the surface electrostatic potential and stability of nucleotide-dendrimer formulations. This approach significantly extends the authors' research on the complexation potential of perfectly branched macromolecules, ultimately explaining previously observed differences and their consequences., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

N 6 -Methyladenosine (m 6 A) represents a common and highly dynamic modification in eukaryotic RNA that affects various cellular pathways. Natural dioxygenases such as FTO and ALKBH5 are enzymes that demethylate m 6 A residues in mRNA. Herein, the first identification of a small-molecule modulator that functions as an artificial m 6 A demethylase is reported. Flavin mononucleotide (FMN), the metabolite produced by riboflavin kinase, mediates substantial photochemical demethylation of m 6 A residues of RNA in live cells. This study provides a new perspective to the understanding of demethylation of m 6 A residues in mRNA and sheds light on the development of powerful small molecules as RNA demethylases and new probes for use in RNA biology., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RNA contains diverse modifications that exert an important influence in a variety of cellular processes. So far, more than 150 modifications have been identified in various RNA species, mainly in ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). In contrast to rRNA, tRNA, and mRNA, the known modifications in small RNA species have been primarily limited to 2'-O-ribose methylation in plants and inosine in mammals. The methylation of small RNAs in mammals is still unclear. Current methods widely used in the characterization of small RNAs are mainly based on the strategy of nucleic acid hybridization and sequencing, which cannot characterize modifications in small RNAs. Herein, we have systematically investigated modifications in small RNAs composed of 16-28 nucleotides (nt) by establishing an effective isolation and neutral enzymatic digestion of small RNAs in combination with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This method allowed us to simultaneously detect 57 different types of nucleoside modification. By using this approach, we revealed 24 modifications in small RNAs comprising 16-28 nt from human cells. In addition, we found that the obesity-associated protein (FTO) may demethylate N 6 -methyladenosine (m 6 A) and N 6 ,2'-O-dimethyladenosine (m 6 Am) in small RNAs of 16-28 nt. Our study demonstrates the existence of diverse modifications in small RNAs composed of 16-28 nt, which may promote in-depth understanding of the regulatory roles of noncoding RNAs., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site. While the N1 of G40 is within 3.4 Å of the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid-base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg 2+ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg 2+ playing a catalytic role in cleavage., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adenosine deaminase converts adenosine into inosine. In contrast to adenosine, relatively little attention has been paid to the physiological roles of inosine. Nevertheless, recent studies have demonstrated that inosine has neuroprotective, cardioprotective, immunomodulatory, and antidepressive effects. Inosine was recently shown to be a less potent agonist than adenosine at the A 2A adenosine receptor. To better depict the differences in the mechanisms of receptor recognition between adenosine and inosine, we carried out supervised molecular dynamics (SuMD) simulations, and the results are analyzed herein., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A description and history of the role that 8-oxo-7,8-dihydroadenine (8-oxoAde) and 8-oxo-7,8-dihydroadenosine (8-oxoA) have in various fields has been compiled. This Review focusses on 1) the formation of this oxidatively generated modification in RNA, its interactions with other biopolymers, and its potential role in the development/progression of disease; 2) the independent synthesis and incorporation of this modified nucleoside into oligonucleotides of RNA to display the progress that has been made in establishing its behavior in biologically relevant systems; 3) reported synthetic routes, which date back to 1890, along with the progress that has been made in the total synthesis of the nucleobase, nucleoside, and their corresponding derivatives; and 4) the isolation, total synthesis, and biological activity of natural products containing these moieties as the backbone. The current state of research regarding this oxidatively generated lesion as well as its importance in the context of RNA, natural products, and potential as drug derivatives is illustrated using all available examples reported to date., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present an innovative O 6 -tert-butyl/N 2 -tert-butyloxycarbonyl protection concept for guanosine (G) phosphoramidites. This concept is advantageous for 2'-modified G building blocks because of very efficient synthetic access when compared with existing routes that usually employ O 6 -(4-nitrophenyl)ethyl/N 2 -acyl protection or that start from 2-aminoadenosine involving enzymatic transformation into guanosine later on in the synthetic path. The new phosphoramidites are fully compatible with 2'-O-tBDMS or TOM phosphoramidites in standard RNA solid-phase synthesis and deprotection, and provide excellent quality of tailored RNAs for the growing range of applications in RNA biophysics, biochemistry, and biology., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

S-Adenosylmethionine (SAM) is an essential methylation cofactor. The origins of SAM methylation are complex, seemingly demanding the simultaneous emergence of an enzyme that makes SAM and enzyme(s) that utilize it. We report that both ATP and adenosine spontaneously react with methionine to yield SAM, thus suggesting that SAM could have emerged by chance. SAM methylation thus exemplifies how metabolites and pathways can co-emerge through the gradual recruitment of individual enzymes in reverse order., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclopropanations, but variants featuring predictable, complementary stereoselectivity in these reactions have remained elusive. In this study, a rationally driven strategy was implemented and applied to engineer myoglobin variants capable of providing access to 1-carboxy-2-aryl-cyclopropanes with high trans-(1R,2R) selectivity and catalytic activity. The stereoselectivity of these cyclopropanation biocatalysts complements that of trans-(1S,2S)-selective variants developed here and previously. In combination with whole-cell biotransformations, these stereocomplementary biocatalysts enabled the multigram synthesis of the chiral cyclopropane core of four drugs (Tranylcypromine, Tasimelteon, Ticagrelor, and a TRPV1 inhibitor) in high yield and with excellent diastereo- and enantioselectivity (98-99.9% de; 96-99.9% ee). These biocatalytic strategies outperform currently available methods to produce these drugs., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand-displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high-throughput single-molecule FRET methods, we compared the kinetics of a putative aptamer-ligand and aptamer-complement strand-displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand-displacement concept can be extended to functional DNA-ligand systems., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Modified adenosine nucleosides have been proposed to be potential DNA-based biomarkers for early diagnosis of tumor and a promising tool for the development of noninvasive prediction systems. However, the low concentration of modified adenosine nucleosides in physiological fluids makes them challenging for both quantitative and qualitative determination. Therefore, materials, which are potentially useful for selective adsorption of nucleobase-containing compounds, were obtained. To obtain the adsorbents, the silica gel particles were coated layer-by-layer with films of the polymers with different combinations of polymers containing thymine groups. Next, the microspheres were irradiated with UV light in the presence of 2'-deoxyadenosine or 5'-deoxy-5'-(methylthio)adenosine, as template molecules, which resulted in the photodimerization of thymine moieties and molecular imprinting of adsorbed modified adenosine compounds. The selectivity of the adsorption was significantly enhanced by the photoimprinting process. Eventually, the imprinted particles have shown an improved ability to recognize mainly 2'-deoxyadenosine and 5'-deoxy-5'-(methylthio)adenosine molecules. The best performing adsorbent was obtained using modified natural polysaccharides. The studied materials could serve as promising adsorbents of biomarkers for tumor diagnostics., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The rationale for the preparation of DNA-templated gold nanoclusters (DNA-Au NCs) has not been well understood, thereby slowing down the advancement of the synthesis and applications of DNA-Au NCs. The interaction between metal ions and the DNA template seems to be the key factor for the successful preparation of DNA-templated metal nanoclusters. With the help of circular dichroism in this contribution, we put efforts into interrogating the necessity of pre-incubation of HAuCl4 with poly-adenine template in the formation of Au NCs by citrate reduction. Our results revealed that the pre-incubation of HAuCl4 with poly-adenine is not favorable for the formation of Au NCs, which is distinctly different from the formation process for silver nanoclusters. It is our hope that this study can provide guidance in the preparation of Au NCs with more DNA templates., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A thermally responsive boronate affinity chromatographic material, which showed thermal sensitivity, had been successfully applied for the enrichment and separation of cis-diol-containing compounds, and the capture and release process could be facilitated by adjusting the temperature. However, in this system, the pH of the mobile phase must be higher than 9.8, and alkaline media can lead to the degradation of labile compounds; the use of silica beads also limits its use. In this study, thermally responsive boronate affinity chromatographic material, namely poly(N-isopropylacrylamide-co-N-acryloyl-3-aminophenylboronic acid) grafted silica, was successfully prepared by atom transfer radical polymerization. Its structure was confirmed by IR spectroscopy and the graft ratio was 20.8%, determined by thermogravimetric analysis. Furthermore, the capture/release of adenosine, a cis-diol, was performed from pH 5.0-9.0 and 10-50°C. The elution of adenosine was remarkably retarded at decreased temperatures and adenosine could be captured completely at 10°C at pH values of 5.0-9.0. The enrichment of adenosine could be achieved by simply changing the temperature from 10 to 50°C. Therefore, this material not only improved the stability of the silica, but was also suitable for the capture of oxidation-sensitive biological analytes. Moreover, it could be used for the enrichment of cis-diol-containing compounds in LC with MS., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although numerous reports on the synthesis of atom-specific (15)N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of (15)N(1) adenosine and (15)N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve (15)N(3) uridine and (15)N(3) cytidine amidites in order to tap full potential of (1)H/(15)N/(15)N-COSY experiments for directly monitoring individual Watson-Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered., (© 2015 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Inhibition of adenosine A2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson's disease (PD). We previously identified the triazolo-9H-purine, ST1535, as a potent A(2A)R antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω-1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω-1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A(2A)R was determined. Two compounds, (2-(3,3-dimethylbutyl)-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-6-amine (3 b) and 4-(6-amino-9-methyl-8-(2H-1,2,3-triazol-2-yl)-9H-purin-2-yl)-2-methylbutan-2-ol (3 c), exhibited good affinity against A(2A)R (Ki =0.4 nM and 2 nM, respectively) and high in vitro metabolic stability (89.5% and 95.3% recovery, respectively, after incubation with HLM for two hours)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

3-Deazaneplanocin A (DzNep) is a potential epigenetic drug for the treatment of various cancers. DzNep has been reported to deplete histone methylations, including oncogenic EZH2 complex, giving rise to epigenetic modifications that reactivate many silenced tumor suppressors in cancer cells. Despite its promise as an anticancer drug, little is known about the structure-activity relationships of DzNep in the context of epigenetic modifications and apoptosis induction. In this study, a number of analogues of DzNep were examined for DzNep-like ability to induce synergistic apoptosis in cancer cells in combination with trichostatin A, a known histone deacetylase (HDAC) inhibitor. The structure-activity relationship data thus obtained provide valuable information on the structural requirements for biological activity. The studies identified three compounds that show similar activities to DzNep. Two of these compounds show good pharmacokinetics and safety profiles. Attempts to correlate the observed synergistic apoptotic activities with measured S-adenosylhomocysteine hydrolase (SAHH) inhibitory activities suggest that the apoptotic activity of DzNep might not be directly due to its inhibition of SAHH., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In atomic force microscopy (AFM) a sharp cantilever tip is used to scan surfaces at the atomic level. One further application is force spectroscopy, in which force-distance curves between binding partners located on the cantilever and substrate surface are determined. This requires specifically immobilized molecules. Herein we describe the covalent binding of single adenosine and thymidine nucleotides on an amino-PEGylated cantilever tip by the phosphoramidite method. Force-distance curves between these cantilever tips and gold surfaces were recorded. The rupture forces of the coordination bond between the primary amine of adenosine and the undercoordinated gold atoms were determined to be 145 pN, which is in agreement with previously published data. The force-distance curves of thymidine-functionalized tips did not show rupture events, because this nucleotide does not possess a primary amine function. Nucleotide-functionalized tips could aid in the understanding of binding mechanisms of nucleotide binding molecules such as polymerases immobilized on surfaces or membranes., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Janus bases are heterocyclic nucleic acid base analogs that present two different faces able to simultaneously hydrogen bond to nucleosides that form Watson-Crick base pairs. The synthesis of a Janus-AT nucleotide analogue, (N)JAT , that has an additional endocyclic ring nitrogen and is thus more capable of efficiently discriminating T/A over G/C bases when base-pairing in a standard duplex-DNA context is described. Conversion to a phosphoramidite ultimately afforded incorporation into an oligonucleotide. In contrast to the first generation of carbocyclic Janus heterocycles, it remains in its unprotonated state at physiological pH and, therefore, forms very stable Watson-Crick base pairs with either A or T bases. Biophysical and computational methods indicate that (N)JAT is an improved candidate for sequence-specific genome targeting., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

3-Deazaneplanocin A (DzNep), a global histone methylation inhibitor, has attracted significant interest in epigenetic research in recent years. The molecular mechanism of action and the cellular off-targets of DzNep, however, are still not well-understood. Our aim was to develop novel DzNep-derived small-molecule probes suitable to be used in live mammalian cells for identification of potential cellular targets of DzNep under physiologically relevant settings. In the current study, we have successfully designed, synthesized, and tested one such probe, called DZ-1. DZ-1 is a 'clickable' affinity-based probe (AfBP) derived from DzNep with minimal structural modifications. The probe was found to be highly cell-permeable, and possessed similar anti-apoptotic activities as DzNep in MCF-7 mammalian cells. Two additional control probes were made as negative labeling/pull-down probes in order to minimize false identification of background proteins due to unavoidable, intrinsic nonspecific photo-crosslinking reactions. All three probes were subsequently used for in-situ proteome profiling in live mammalian cells, followed by large-scale pull-down/LC-MS/MS analysis for identification of potential cellular protein targets that might interact with DzNep in native cellular environments. Our LC-MS/MS results revealed some highly enriched proteins that had not been reported as potential DzNep targets. These proteins might constitute unknown cellular off-targets of DzNep. Though further validation experiments are needed in order to unequivocally confirm these off-targets, our findings shed new light on the future use of DzNep as a validated chemical probe for epigenetic research and as a potential drug candidate for cancer therapy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The intrinsic acid-base properties of the hexa-2'-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT) [=(A1∙G2∙G3∙C4∙C5∙T6)=(HNPP)⁵⁻] have been determined by ¹H NMR shift experiments. The pKa values of the individual sites of the adenosine (A), guanosine (G), cytidine (C), and thymidine (T) residues were measured in water under single-strand conditions (i.e., 10% D₂O, 47 °C, I=0.1 M, NaClO₄). These results quantify the release of H⁺ from the two (N7)H⁺ (G∙G), the two (N3)H⁺ (C∙C), and the (N1)H⁺ (A) units, as well as from the two (N1)H (G∙G) and the (N3)H (T) sites. Based on measurements with 2'-deoxynucleosides at 25 °C and 47 °C, they were transferred to pKa values valid in water at 25 °C and I=0.1 M. Intramolecular stacks between the nucleobases A1 and G2 as well as most likely also between G2 and G3 are formed. For HNPP three pKa clusters occur, that is those encompassing the pKa values of 2.44, 2.97, and 3.71 of G2(N7)H⁺, G3(N7)H⁺, and A1(N1)H⁺, respectively, with overlapping buffer regions. The tautomer populations were estimated, giving for the release of a single proton from five-fold protonated H₅(HNPP)(±) , the tautomers (G2)N7, (G3)N7, and (A1)N1 with formation degrees of about 74, 22, and 4%, respectively. Tautomer distributions reveal pathways for proton-donating as well as for proton-accepting reactions both being expected to be fast and to occur practically at no "cost". The eight pKa values for H₅(HNPP)(±) are compared with data for nucleosides and nucleotides, revealing that the nucleoside residues are in part affected very differently by their neighbors. In addition, the intrinsic acidity constants for the RNA derivative r(A1∙G2∙G3∙C4∙C5∙U6), where U=uridine, were calculated. Finally, the effect of metal ions on the pKa values of nucleobase sites is briefly discussed because in this way deprotonation reactions can easily be shifted to the physiological pH range., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Writer's blocks: The first synthesis of RNA purine building blocks, 4'-selenoadenosine and 4'-selenoguanosine was achieved from D-ribose by regioisomeric rearrangement, which was confirmed by X-ray crystallography. 4'-Selenoadenosine exists in an unusual mixture of north and south conformers in the solid state., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The route taken by a DNA cargo on a branched track can be controlled by the small molecule adenosine using a pair of aptamers that reciprocally block and unblock branches of the track in response to adenosine binding., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A concise synthesis of a series of N(6)-substituted adenosines with bicyclo[3.2.1]octan-6-yl and polycyclic N(6)-substituents has been developed. The adenosine A(1) receptor (A(1)R) affinity and potency of these compounds was initially assessed using competitive binding assays and cyclic adenosine monophosphate (cAMP) accumulation assays in DDT(1) MF-2 cells. The potency and receptor subtype selectivity of selected examples was further evaluated by measuring their effects on cAMP accumulation at all human adenosine receptor subtypes expressed in CHO cells. The results of these assays indicated that all of the synthesised N(6)-substituted adenosines are full agonists at A(1) R and activate this receptor selectively over the other adenosine receptor subtypes. The two standout compounds in terms of potency were N(6)-(3-thiabicyclo[3.2.1]octan-6-yl)adenosine and N(6)-(cubanylmethyl)adenosine with EC(50) values at human A(1)R of 2.3 nM and 1.1 nM, respectively. The cubanylmethyl derivative in particular proved to be highly receptor subtype selective. These two compounds were further evaluated in a simulated ischaemia model in cultured cardiomyoblasts, where they were found to impart protective effects under hypoxic conditions that resulted in a significant reduction in cell death., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel compound, N⁶-(4-hydroxybenzyl)adenosine, isolated from Gastrodia elata and which has been shown to be a potential therapeutic agent for preventing and treating neurodegenerative disease, was found to target both the adenosine A(2A) receptor (A(2A) R) and the equilibrative nucleoside transporter 1 (ENT1). As A(2A) R and ENT1 are proximal in the synaptic crevice of striatum, where the mutant huntingtin aggregate is located, the dual-action compounds that concomitantly target these two membrane proteins may be beneficial for the therapy of Huntington's disease. To design the desired dual-action compounds, pharmacophore models of the A(2A) R agonists and the ENT1 inhibitors were constructed. Accordingly, potentially active compounds were designed and synthesized by chemical modification of adenosine, particularly at the N⁶ and C⁵' positions, if the predicted activity was within an acceptable range. Indeed, some of the designed compounds exhibit significant dual-action properties toward both A(2A) R and ENT1. Both pharmacophore models exhibit good statistical correlation between predicted and measured activities. In agreement with competitive ligand binding assay results, these compounds also prevent apoptosis in serum-deprived PC12 cells, rendering a crucial function in neuroprotection and potential utility in the treatment of neurodegenerative diseases., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adenosine (Ado) can accept three protons, at N1, N3, and N7, to give H(3) (Ado)(3+) , and thus has three macro acidity constants. Unfortunately, these constants do not reflect the real basicity of the N sites due to internal repulsions, for example, between (N1)H(+) and (N7)H(+). However, these macroconstants are still needed for the evaluations and the first two are taken from our own earlier work, that is, pK(H)(H(3))((Ado)) = -4.02 and pK(H)(H(2))((Ado)) = -1.53; the third one was re-measured as pK(H)(H)((Ado)) = 3.64 ± 0.02 (25 °C; I=0.5 M, NaNO(3)), because it is the main basis for evaluating the intrinsic basicities of N7 and N3. Previously, contradicting results had been published for the micro acidity constant of the (N7)H(+) site; this constant has now been determined in an unequivocal manner, and that of the (N3)H(+) site was obtained for the first time. The micro acidity constants, which describe the release of a proton from an (N)H(+) site under conditions for which the other nitrogen atoms are free and do not carry a proton, decrease in the order pk(N7-N1)(N7(Ado)N1·H)) = 3.63 ± 0.02 > pk(N7-N1)(H·N7(Ado)N1) = 2.15 ± 0.15 > pk(N3-N1,N7)(H·N3(Ado)N1,N7) =1.5 ± 0.3, reflecting the decreasing basicity of the various nitrogen atoms, that is, N1>N7>N3. Application of the above-mentioned microconstants allows one to calculate the percentages (formation degrees) of the tautomers formed for monoprotonated adenosine, H(Ado)(+) , in aqueous solution; the results are 96.1, 3.2, and 0.7% for N7(Ado)N1·H(+), (+)H·N7(Ado)N1, and (+)H·N3(Ado)N1,N7, respectively. These results are in excellent agreement with theoretical DFT calculations. Evidently, H(Ado)(+) exists to the largest part as N7(Ado)N1·H(+) having the proton located at N1; the two other tautomers are minority species, but they still form. These results are not only meaningful for adenosine itself, but are also of relevance for nucleic acids and adenine nucleotides, as they help to understand their metal ion-binding properties; these aspects are briefly discussed., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)