Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies.

Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.

NME6: ribonucleotide salvage sustains mitochondrial transcription.

The building blocks for RNA and DNA are made in the cytosol, meaning mitochondria depend on the import and salvage of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) for the synthesis of their own genetic material. While extensive research has focused on mitochondrial dNTP homeostasis due to its defects being associated with various mitochondrial DNA (mtDNA) depletion and deletion syndromes, the investigation of mitochondrial rNTP homeostasis has received relatively little attention. In this issue of the EMBO Journal, Grotehans et al provide compelling evidence of a major role for NME6, a mitochondrial nucleoside diphosphate kinase, in the conversion of pyrimidine ribonucleoside diphosphates into the corresponding triphosphates. These data also suggest a significant physiological role for NME6, as its absence results in the depletion of mitochondrial transcripts and destabilization of the electron transport chain (Grotehans et al, 2023).

Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Optimized infrared photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (IR-PAR-CLIP) protocol identifies novel IGF2BP3-interacting RNAs in colon cancer cells.

The conserved family of RNA-binding proteins (RBPs), IGF2BPs, plays an essential role in posttranscriptional regulation controlling mRNA stability, localization, and translation. Mammalian cells express three isoforms of IGF2BPs: IGF2BP1-3. IGF2BP3 is highly overexpressed in cancer cells, and its expression correlates with a poor prognosis in various tumors. Therefore, revealing its target RNAs with high specificity in healthy tissues and in cancer cells is of crucial importance. Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies the binding sites of RBPs on their target RNAs at nucleotide resolution in a transcriptome-wide manner. Here, we optimized the PAR-CLIP protocol to study RNA targets of endogenous IGF2BP3 in a human colorectal carcinoma cell line. To this end, we first established an immunoprecipitation protocol to obtain highly pure endogenous IGF2BP3-RNA complexes. Second, we modified the protocol to use highly sensitive infrared (IR) fluorescent dyes instead of radioactive probes to visualize IGF2BP3-crosslinked RNAs. We named the modified method "IR-PAR-CLIP." Third, we compared RNase cleavage conditions and found that sequence preferences of the RNases impact the number of the identified IGF2BP3 targets and introduce a systematic bias in the identified RNA motifs. Fourth, we adapted the single adapter circular ligation approach to increase the efficiency in library preparation. The optimized IR-PAR-CLIP protocol revealed novel RNA targets of IGF2BP3 in a human colorectal carcinoma cell line. We anticipate that our IR-PAR-CLIP approach provides a framework for studies of other RBPs.

Drug Resistance Assessed in a Phase 3 Clinical Trial of Maribavir Therapy for Refractory or Resistant Cytomegalovirus Infection in Transplant Recipients.

BACKGROUND: This drug resistance analysis of a randomized trial includes 234 patients receiving maribavir and 116 receiving investigator-assigned standard therapy (IAT), where 56% and 24%, respectively, cleared cytomegalovirus DNA at week 8 (treatment responders). METHODS: Baseline and posttreatment plasma samples were tested for mutations conferring drug resistance in viral genes UL97, UL54, and UL27. RESULTS: At baseline, genotypic testing revealed resistance to ganciclovir, foscarnet, or cidofovir in 56% of patients receiving maribavir and 68% receiving IAT, including 9 newly phenotyped mutations. Among them, 63% (maribavir) and 21% (IAT) were treatment responders. Detected baseline maribavir resistance mutations were UL27 L193F (n = 1) and UL97 F342Y (n = 3). Posttreatment, emergent maribavir resistance mutations were detected in 60 (26%) of those randomized to maribavir, including 49 (48%) of 103 nonresponders and 25 (86%) of the 29 nonresponders where viral DNA initially cleared then rebounded while on maribavir. The most common maribavir resistance mutations were UL97 T409M (n = 34), H411Y (n = 26), and C480F (n = 21), first detected 26 to 130 (median 56) days after starting maribavir. CONCLUSIONS: Baseline maribavir resistance was rare. Drug resistance to standard cytomegalovirus antivirals did not preclude treatment response to maribavir. Rebound in plasma cytomegalovirus DNA while on maribavir strongly suggests emerging drug resistance. CLINICAL TRIALS REGISTRATION: NCT02931539.

Cost-effectiveness of maribavir versus conventional antiviral therapies for post-transplant refractory cytomegalovirus infection with or without genotypic resistance: A US perspective.

This study evaluated the cost-effectiveness of maribavir versus investigator-assigned therapy (IAT; valganciclovir/ganciclovir, foscarnet, or cidofovir) for post-transplant refractory cytomegalovirus (CMV) infection with or without resistance. A two-stage Markov model was designed using data from the SOLSTICE trial (NCT02931539), real-world multinational observational studies, and published literature. Stage 1 (0-78 weeks) comprised clinically significant CMV (csCMV), non-clinically significant CMV (n-csCMV), and dead states; stage 2 (78 weeks-lifetime) comprised alive and dead states. Total costs (2022 USD) and quality-adjusted life years (QALYs) were estimated for the maribavir and IAT cohorts. An incremental cost-effectiveness ratio was calculated to determine cost-effectiveness against a willingness-to-pay threshold of $100 000/QALY. Compared with IAT, maribavir had lower costs ($139 751 vs $147 949) and greater QALYs (6.04 vs 5.83), making it cost-saving and more cost-effective. Maribavir had higher acquisition costs compared with IAT ($80 531 vs $65 285), but lower costs associated with administration/monitoring ($16 493 vs $27 563), adverse events (AEs) ($11 055 vs $16 114), hospitalization ($27 157 vs $33 905), and graft loss ($4516 vs $5081), thus making treatment with maribavir cost-saving. Maribavir-treated patients spent more time without CMV compared with IAT-treated patients (0.85 years vs 0.68 years), leading to lower retreatment costs for maribavir (cost savings: -$42 970.80). Compared with IAT, maribavir was more cost-effective for transplant recipients with refractory CMV, owing to better clinical efficacy and avoidance of high costs associated with administration, monitoring, AEs, and hospitalizations. These results can inform healthcare decision-makers on the most effective use of their resources for post-transplant refractory CMV treatment.

Spectral libraries from nucleobases and deoxyribonucleosides facilitate the identification of ribonucleosides by nano-flow liquid chromatography-tandem mass spectrometry.

RATIONALE: The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable. METHODS: Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Spectral libraries were constructed from homologous nucleobases and deoxyribonucleosides using targeted MS2 and neutral-loss-triggered MS3 methods, and collision energies were optimized. The feasibility of using these libraries for identifying modified ribonucleosides and their positional isomers was assessed through comparison of spectral fragmentation patterns. RESULTS: Our analysis reveals that both MS2 and neutral-loss-triggered MS3 methods yielded rich spectra with similar fragmentation patterns across ribonucleosides, deoxyribonucleosides and nucleobases. Moreover, we demonstrate that spectra from nucleobases and deoxyribonucleosides, generated at optimized collision energies, exhibited sufficient similarity to those of modified ribonucleosides to enable their use as reference spectra for accurate identification of positional isomers within ribonucleoside families. CONCLUSIONS: The study demonstrates the efficacy of utilizing homologous nucleobases and deoxyribonucleosides as interchangeable reference spectral libraries for identifying modified ribonucleosides and their positional isomers. This approach offers a valuable solution for overcoming limitations posed by the unavailability of commercial standards, enhancing the analysis of RNA post-transcriptional modifications via mass spectrometry.

RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine.

Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.

Cytomegalovirus related hospitalization costs among hematopoietic stem cell and solid organ transplant recipients treated with maribavir versus investigator-assigned therapy: A US-based study.

BACKGROUND: Cytomegalovirus (CMV) infections among hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients impose a significant health care resource utilization (HCRU)-related economic burden. Maribavir (MBV), a novel anti-viral therapy (AVT), approved by the United States Food and Drug Administration for post-transplant CMV infections refractory (with/without resistance) to conventional AVTs has demonstrated lower hospital length of stay (LOS) versus investigator-assigned therapy (IAT; valgancilovir, ganciclovir, foscarnet, or cidofovir) in a phase 3 trial (SOLSTICE). This study estimated the HCRU costs of MBV versus IAT. METHODS: An economic model was developed to estimate HCRU costs for patients treated with MBV or IAT. Mean per-patient-per-year (PPPY) HCRU costs were calculated using (i) annualized mean hospital LOS in SOLSTICE, and (ii) CMV-related direct costs from published literature. Probabilistic sensitivity analysis with Monte-Carlo simulations assessed model robustness. RESULTS: Of 352 randomized patients receiving MBV (n = 235) or IAT (n = 117) for 8 weeks in SOLSTICE, 40% had HSCT and 60% had SOT. Mean overall PPPY HCRU costs of overall hospital-LOS were $67,205 (95% confidence interval [CI]: $33,767, $231,275) versus $145,501 (95% CI: $62,064, $589,505) for MBV and IAT groups, respectively. Mean PPPY ICU and non-ICU stay costs were: $32,231 (95% CI: $5,248, $184,524) versus $45,307 (95% CI: $3,957, $481,740) for MBV and IAT groups, and $82,237 (95% CI: $40,397, $156,945) MBV versus $228,329 (95% CI: $94,442, $517,476) for MBV and IAT groups, respectively. MBV demonstrated cost savings in over 99.99% of simulations. CONCLUSIONS: This analysis suggests that Mean PPPY HCRU costs were 29%-64% lower with MBV versus other-AVTs.

Use of Maribavir for Multidrug Resistant Cytomegaloviremia in a Pediatric Oncology Patient.

Resistant and refractory cytomegalovirus (CMV) viremia can limit the provision of chemotherapy due to myelosuppression and end-organ dysfunction. Few therapies are available for children with clinically significant CMV viremia. We successfully used maribavir for a 4-year-old patient with lymphoma to complete his chemotherapy course. Resistance to maribavir did result after many months of therapy.

Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer.

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current-based MS1 workflow to quantify ∼6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and 'drug response' mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.

Towards a comprehensive understanding of RNA deamination: synthesis and properties of xanthosine-modified RNA.

Nucleobase deamination, such as A-to-I editing, represents an important posttranscriptional modification of RNA. When deamination affects guanosines, a xanthosine (X) containing RNA is generated. However, the biological significance and chemical consequences on RNA are poorly understood. We present a comprehensive study on the preparation and biophysical properties of X-modified RNA. Thermodynamic analyses revealed that base pairing strength is reduced to a level similar to that observed for a G•U replacement. Applying NMR spectroscopy and X-ray crystallography, we demonstrate that X can form distinct wobble geometries with uridine depending on the sequence context. In contrast, X pairing with cytidine occurs either through wobble geometry involving protonated C or in Watson-Crick-like arrangement. This indicates that the different pairing modes are of comparable stability separated by low energetic barriers for switching. Furthermore, we demonstrate that the flexible pairing properties directly affect the recognition of X-modified RNA by reverse transcription enzymes. Primer extension assays and PCR-based sequencing analysis reveal that X is preferentially read as G or A and that the ratio depends on the type of reverse transcriptase. Taken together, our results elucidate important properties of X-modified RNA paving the way for future studies on its biological significance.

MODOMICS: a database of RNA modification pathways. 2021 update.

The MODOMICS database has been, since 2006, a manually curated and centralized resource, storing and distributing comprehensive information about modified ribonucleosides. Originally, it only contained data on the chemical structures of modified ribonucleosides, their biosynthetic pathways, the location of modified residues in RNA sequences, and RNA-modifying enzymes. Over the years, prompted by the accumulation of new knowledge and new types of data, it has been updated with new information and functionalities. In this new release, we have created a catalog of RNA modifications linked to human diseases, e.g., due to mutations in genes encoding modification enzymes. MODOMICS has been linked extensively to RCSB Protein Data Bank, and sequences of experimentally determined RNA structures with modified residues have been added. This expansion was accompanied by including nucleotide 5'-monophosphate residues. We redesigned the web interface and upgraded the database backend. In addition, a search engine for chemically similar modified residues has been included that can be queried by SMILES codes or by drawing chemical molecules. Finally, previously available datasets of modified residues, biosynthetic pathways, and RNA-modifying enzymes have been updated. Overall, we provide users with a new, enhanced, and restyled tool for research on RNA modification. MODOMICS is available at https://iimcb.genesilico.pl/modomics/.

Elevated Plasma Concentration of 4-Pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) Highlights Malignancy of Renal Cell Carcinoma.

Nicotinamide (NA) derivatives play crucial roles in various biological processes, such as inflammation, regulation of the cell cycle, and DNA repair. Recently, we proposed that 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR), an unusual derivative of NA, could be classified as an oncometabolite in bladder, breast, and lung cancer. In this study, we investigated the relations between NA metabolism and the progression, recurrence, metastasis, and survival of patients diagnosed with different histological subtypes of renal cell carcinoma (RCC). We identified alterations in plasma NA metabolism, particularly in the clear cell RCC (ccRCC) subtype, compared to papillary RCC, chromophobe RCC, and oncocytoma. Patients with ccRCC also exhibited larger tumor sizes and elevated levels of diagnostic serum biomarkers, such as hsCRP concentration and ALP activity, which were positively correlated with the plasma 4PYR. Notably, 4PYR levels were elevated in advanced stages of ccRCC cancer and were associated with a highly aggressive phenotype of ccRCC. Additionally, elevated concentrations of 4PYR were related to a higher likelihood of mortality, recurrence, and particularly metastasis in ccRCC. These findings are consistent with other studies, suggesting that NA metabolism is accelerated in RCC, leading to abnormal concentrations of 4PYR. This supports the concept of 4PYR as an oncometabolite and a potential prognostic factor in the ccRCC subtype.

Mizoribine Promotes Molecular Chaperone HSP60/HSP10 Complex Formation.

It has been reported that Mizoribine is an immunosuppressant used to suppress rejection in renal transplantation, nephrotic syndrome, lupus nephritis, and rheumatoid arthritis. The molecular chaperone HSP60 alone induces inflammatory cytokine IL-6 and the co-chaperone HSP10 alone inhibits IL-6 induction. HSP60 and HSP10 form a complex in the presence of ATP. We analyzed the effects of Mizoribine, which is structurally similar to ATP, on the structure and physiological functions of HSP60-HSP10 using Native/PAGE and transmission electron microscopy. At low concentrations of Mizoribine, no complex formation of HSP60-HSP10 was observed, nor was the expression of IL-6 affected. On the other hand, high concentrations of Mizoribine promoted HSP60-HSP10 complex formation and consequently suppressed IL-6 expression. Here, we propose a novel mechanism of immunosuppressive action of Mizoribine.

1H-1,2,3-triazolyl-1,6-naphthyridin-7(6H)-ones as Potential Fluorescent Nucleoside Analogues: Synthesis and Optical Properties.

In this article, we present the synthesis and the optical properties of three original molecules as potential fluorescent ribonucleoside analogues incorporating a 1,6-naphthyridin-7(6H)-one scaffold as a fluorescent nucleobase and a 1,2,3-triazole as a linkage. The nucleosides were prepared via a Cu alkyne-azide cycloaddition (CuAAC) reaction between a ribofuranosyl azide and a 4-ethynylpyridine partner. Construction of substituted 1,6-naphthyridin-7(6H)-ones was achieved through two additional steps. Optical property studies were investigated on nucleoside analogues. Powerful fluorescence properties have been evidenced with a remarkable change of emissivity depending on the polarity of the solvent, making these molecules suitable as a new class of artificial fluorescent nucleosides for investigating enzyme binding sites as well as probing nucleic acids. In addition, we are convinced that such analogues could be of great interest in the search for new antiviral or antitumoral drugs based on nucleosides.

Selective Inhibition of ADAR1 Using 8-Azanebularine-Modified RNA Duplexes.

Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1's role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain's duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5' and 8 bp 3' to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.

Enhancing Prebiotic Phosphorylation and Modulating the Regioselectivity of Nucleosides with Diamidophosphate†.

Among the many prebiotic phosphorylation chemistries investigated, diamidophosphate (DAP) has shown promising potential for nucleoside phosphorylation. Herein, we show that DAP's phosphorylation capability is enhanced significantly (up to 90%) in wet-dry cycles by a range of prebiotically plausible pHs (6-10) and temperatures (up to 80 °C) in the presence of additives such as formamide, cyanamide, urea, guanidine, 2-aminoimidazole, and hydantoin. For ribonucleosides, the main products are the 2',3'-cyclic phosphates along with the corresponding 2'- and 3'-phosphates, while deoxyribonucleosides form 5'- and 3'-phosphates, the ratios of which are affected by cycles and the presence and nature of the additives. A simple change of temperature to 80 °C with additives leads to higher conversion yields (≈80-90%) with an increased level of 5'-phosphorylation (≈40-49%). This demonstration of enhancing and controlling the regioselectivity of DAP-mediated phosphorylation by a range of additives and conditions potentiates transitioning to the search for more efficient catalysts, enabling regiospecific phosphorylations and oligonucleotide formation in the same milieu and setting.

Oligonucleotide Catabolism-Derived Gluconucleosides in Caenorhabditis elegans.

Nucleosides are essential cornerstones of life, and nucleoside derivatives and synthetic analogues have important biomedical applications. Correspondingly, production of non-canonical nucleoside derivatives in animal model systems is of particular interest. Here, we report the discovery of diverse glucose-based nucleosides in Caenorhabditis elegans and related nematodes. Using a mass spectrometric screen based on all-ion fragmentation in combination with total synthesis, we show that C. elegans selectively glucosylates a series of modified purines but not the canonical purine and pyrimidine bases. Analogous to ribonucleosides, the resulting gluconucleosides exist as phosphorylated and non-phosphorylated forms. The phosphorylated gluconucleosides can be additionally decorated with diverse acyl moieties from amino acid catabolism. Syntheses of representative variants, facilitated by a novel 2'-O- to 3'-O-dibenzyl phosphoryl transesterification reaction, demonstrated selective incorporation of different nucleobases and acyl moieties. Using stable-isotope labeling, we further show that gluconucleosides incorporate modified nucleobases derived from RNA and possibly DNA breakdown, revealing extensive recycling of oligonucleotide catabolites. Gluconucleosides are conserved in other nematodes, and biosynthesis of specific subsets is increased in germline mutants and during aging. Bioassays indicate that gluconucleosides may function in stress response pathways.