Enhancement of Cordycepin Production through Liquid Static Fermentation of Caterpillar Medicinal Mushroom Cordyceps militaris (Ascomycetes).

Cordycepin (3'-deoxyadenosine) is a bioactive nucleoside analog synthesized by Cordyceps militaris. Liquid fermentation of C. militaris by addition in different concentrations of five additives singly was evaluated. Glycine at 15.00 g/L after 20 d enhanced the cordycepin of 1773.33 mg/L (15-fold increment over control). Adenine at 4.00 g/L and 6.00 g/L in the liquid media showed significantly higher cordycepin i.e.1596.66 mg/L and 1550.00 mg/L (3-fold increment over control) after 40 d. Tryptone supplementation 14.00 g/L significantly higher cordycepin 784.33 mg/L (6.70-fold increment over control) and 912.66 mg/L production after 20 and 40 d of inoculation. Peanut oil at 10.00 g/L produced 585.66 mg/L (5-fold increment over control) cordycepin after 20 d and after 40 d, also addition of peanut oil at 20.00 g/L and 30.00 g/L in the media showed 631.66 and 624.31 mg/L cordycepin content. Supplementation of mono-sodium glutamate at 0.30 g/L produced significantly highest cordycepin i.e. 614 mg/L and 635.00 mg/L cordycepin after 20 and 40 d, respectively.

Role of 6mA in the Regulation of Metabolic Biosynthesis in Sorghum Callus.

Plant cell culture technology helps to obtain natural plant-derived metabolites. The callus of sorghum, a prominent cereal crop, possesses various metabolites with potential health benefits. However, the epigenetic mechanism regulating metabolic biosynthetic capabilities in sorghum remains unknown. Therefore, we conducted N6-methyladenine (6mA) methylome analysis using transcriptome profiling and metabolome analysis to investigate the role of 6mA alterations in two calluses having different biosynthetic capacities, which were derived from immature sorghum embryos. Our findings indicate that the 6mA upregulation within gene bodies is crucial in transcriptional activity potentially mediated by the DNA demethylase SbALKBH1. Furthermore, 6mA was significantly enriched in genes involved in the biosynthesis of flavonoids and isoflavonoids. This could serve as a novel source of bioactive compounds for human health. Thus, 6mA could play an essential role in flavonoid biosynthesis in the sorghum callus.

Symmetric and asymmetric DNA N6-adenine methylation regulates different biological responses in Mucorales.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model.

Duchenne muscular dystrophy (DMD) affecting 1 in 3500-5000 live male newborns is the frequently fatal genetic disease resulted from various mutations in DMD gene encoding dystrophin protein. About 70% of DMD-causing mutations are exon deletion leading to frameshift of open reading frame and dystrophin deficiency. To facilitate translating human DMD-targeting CRISPR therapeutics into patients, we herein establish a genetically humanized mouse model of DMD by replacing exon 50 and 51 of mouse Dmd gene with human exon 50 sequence. This humanized mouse model recapitulats patient's DMD phenotypes of dystrophin deficiency and muscle dysfunction. Furthermore, we target splicing sites in human exon 50 with adenine base editor to induce exon skipping and robustly restored dystrophin expression in heart, tibialis anterior and diaphragm muscles. Importantly, systemic delivery of base editor via adeno-associated virus in the humanized male mouse model improves the muscle function of DMD mice to the similar level of wildtype ones, indicating the therapeutic efficacy of base editing strategy in treating most of DMD types with exon deletion or point mutations via exon-skipping induction.

Insights into the A-C Mismatch Conformational Ensemble in Duplex DNA and its Role in Genetic Processes through a Structure-based Review.

Knowing the conformational ensembles formed by mismatches is crucial for understanding how they are generated and repaired and how they contribute to genomic instability. Here, we review structural and energetic studies of the A-C mismatch in duplex DNA and use the information to identify critical conformational states in its ensemble and their significance in genetic processes. In the 1970s, Topal and Fresco proposed the A-C wobble stabilized by two hydrogen bonds, one requiring protonation of adenine-N1. Subsequent NMR and X-ray crystallography studies showed that the protonated A-C wobble was in dynamic equilibrium with a neutral inverted wobble. The mismatch was shown to destabilize duplex DNA in a sequence- and pH-dependent manner by 2.4-3.8 kcal/mol and to have an apparent pKa ranging between 7.2 and 7.7. The A-C mismatch conformational repertoire expanded as structures were determined for damaged and protein-bound DNA. These structures included Watson-Crick-like conformations forming through tautomerization of the bases that drive replication errors, the reverse wobble forming through rotation of the entire nucleotide proposed to increase the fidelity of DNA replication, and the Hoogsteen base-pair forming through the flipping of the adenine base which explained the unusual specificity of DNA polymerases that bypass DNA damage. Thus, the A-C mismatch ensemble encompasses various conformational states that can be selectively stabilized in response to environmental changes such as pH shifts, intermolecular interactions, and chemical modifications, and these adaptations facilitate critical biological processes. This review also highlights the utility of existing 3D structures to build ensemble models for nucleic acid motifs.

Enterococcus faecalis-derived adenine enhances enterohaemorrhagic Escherichia coli Type 3 Secretion System-dependent virulence.

Interactions between microbiota and enteric pathogens can promote colonization resistance or enhance pathogenesis. The pathobiont Enterococcus faecalis increases enterohaemorrhagic E. coli (EHEC) virulence by upregulating Type 3 Secretion System (T3SS) expression, effector translocation, and attaching and effacing (AE) lesion formation on enterocytes, but the mechanisms underlying this remain unknown. Using co-infection of organoids, metabolomics, supplementation experiments and bacterial genetics, here we show that co-culture of EHEC with E. faecalis increases the xanthine-hypoxanthine pathway activity and adenine biosynthesis. Adenine or E. faecalis promoted T3SS gene expression, while transcriptomics showed upregulation of adeP expression, which encodes an adenine importer. Mechanistically, adenine relieved High hemolysin activity (Hha)-dependent repression of T3SS gene expression in EHEC and promoted AE lesion formation in an AdeP-dependent manner. Microbiota-derived purines, such as adenine, support multiple beneficial host responses; however, our data show that this metabolite also increases EHEC virulence, highlighting the complexity of pathogen-microbiota-host interactions in the gut.

ABE-ultramax for high-efficiency biallelic adenine base editing in zebrafish.

Advancements in CRISPR technology, particularly the development of base editors, revolutionize genetic variant research. When combined with model organisms like zebrafish, base editors significantly accelerate and refine in vivo analysis of genetic variations. However, base editors are restricted by protospacer adjacent motif (PAM) sequences and specific editing windows, hindering their applicability to a broad spectrum of genetic variants. Additionally, base editors can introduce unintended mutations and often exhibit reduced efficiency in living organisms compared to cultured cell lines. Here, we engineer a suite of adenine base editors (ABEs) called ABE-Ultramax (Umax), demonstrating high editing efficiency and low rates of insertions and deletions (indels) in zebrafish. The ABE-Umax suite of editors includes ABEs with shifted, narrowed, or broadened editing windows, reduced bystander mutation frequency, and highly flexible PAM sequence requirements. These advancements have the potential to address previous challenges in disease modeling and advance gene therapy applications.

Unexpected large charge transfer rate mediated by adenine in twisted DNA structure.

DNA exhibits remarkable charge transfer ability, which is crucial for its biological functions and potential electronic applications. The charge transfer process in DNA is widely recognized as primarily mediated by guanine, while the contribution of other nucleobases is negligible. Using the tight-binding models in conjunction with first-principles calculations, we investigated the charge transfer behavior of homogeneous GC and AT pairs. We found that the charge transfer rate of adenine significantly changes. With overstretching, the charge transfer rate of adenine can even surpass that of guanine, by as much as five orders of magnitude at a twist angle of around 26°. Further analysis reveals that it is attributed to the turnover of the relative coupling strength between homogeneous GC and AT base pairs, which is caused by the symmetry exchange between the two highest occupied molecular orbitals of base pairs occurring at different twist angles. Given the high degree of flexibility of DNA in vivo and in vitro conditions, these findings prompt us to reconsider the mechanism of biological functions concerning the charge transfer in DNA molecules and further open the potential of DNA as a biomaterial for electronic applications.

Insights on multi-spectroscopic approaches for estimating the in vitro binding of favipiravir with adenine nucleotide.

Favipiravir (FVP) is an oral antiviral drug approved in 2021 for the treatment of COVID-19. It is a pyrazine derivative that can be integrated into anti-viral RNA products to inhibit viral replication. While, adenine is a purine nucleobase that is found in deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to generate genetic information. For the first time, the binding mechanism between FVP and adenine was determined using different techniques, including UV-visible spectrophotometry, spectrofluorimetry, synchronous fluorescence (SF) spectroscopy, Fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET), and metal ion complexation. The fluorescence spectra indicated that FVP is bound to adenine via Van der Waals forces and hydrogen bonding through a spontaneous binding process (ΔGο < 0). The quenching mechanism was found to be static. Various temperature settings were used to investigate thermodynamic characteristics, such as binding forces, binding constants, and the number of binding sites. The reaction parameters, including the enthalpy change (ΔHο) and entropy change (ΔSο), were calculated using Van't Hoff's equation. The findings demonstrated that the adenine-FVP binding was endothermic. Furthermore, the results of the experiments revealed that some metal ions (K+, Ca+2, Co+2, Cu+2, and Al+3) might facilitate the binding interaction between FVP and adenine. Slight changes are observed in the FTIR spectra of adenine, indicating the binding interaction between adenine and FVP. This study may be useful in understanding the pharmacokinetic characteristics of FVP and how the drug binds to adenine to prevent any side effects.

De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants.

Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles in OsALS and creating synonymous mutations in OsSPL14 to resist OsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox.

Library-Assisted Evolution in Eukaryotic Cells Yield Adenine Base Editors with Enhanced Editing Specificity.

The current-generation adenine base editor (ABE) ABE8e, which has evolved from the prokaryotic evolution system, exhibits high efficiency in mediating A-to-G conversion and is presumed to be promising for gene therapy. However, its much wider editing window and substantially higher off-target editing activity restricted its applications in precise base editing for therapeutic use. This study uses a library-assisted protein evolution approach using eukaryotic cells to generate ABE variants with improved specificity and reduced off-target editing while maintaining high activity in human cells. The study generated an expanded set of ABEs with efficient editing activities and chose four evolved variants that offered either similar or modestly higher efficiency within a narrower editing window of protospacer position ≈4-7 compared to that of ABE8e in human cells, which would enable minimized bystander editing. Moreover, these variants resulted in reduced off-target editing events when delivered as plasmid or mRNA into human cells. Finally, these variants can install both disease-suppressing mutations and disease-correcting mutations efficiently with minimal undesired bystander editing making them promising approaches for specific therapeutic edits. In summary, the work establishes a mutant-library-assisted protein evolution method in eukaryotic cells and generates alternative ABE variants as efficient tools for precise human genome editing.

Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e.

Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.

Isomerization Pathways of a Mismatched Base Pair of A:8OG in Free Duplex DNA.

The A:8OG base pair (bp) is the outcome of DNA replication of the mismatched C:8OG bp. A high A:8OG bp population increases the C/G to A/T transversion mutation, which is responsible for various diseases. MutY is an important enzyme in the error-proof cycle and reverts A:8OG to C:8OG bp by cleaving adenine from the A:8OG bp. Several X-ray crystallography studies have determined the structure of MutY during the lesion scanning and lesion recognition stages. Interestingly, glycosidic bond (χ) angles of A:8OG bp in those two lesion recognition structures were found to differ, which implies that χ-torsion isomerization should occur during the lesion recognition process. In this study, as a first step to understanding this isomerization process, we characterized the intrinsic dynamic features of A:8OG in free DNAs by a free energy landscape simulation at the all-atom level. In this study, four isomerization states were assigned in the order of abundance: Aanti:8OGsyn > Aanti:8OGanti > Asyn:8OGanti ≈ Asyn:8OGsyn. Of these bp states, only 8OG in Asyn:8OGanti was located in the extrahelical space, whereas the purine bases (A and 8OG) in the other bp states remained inside the DNA helix. Also, free energy landscapes showed that the isomerization processes connecting these four bp states proceeded mostly in the intrahelical space via successive single glycosidic bond rotations of either A or 8OG.

Structural basis for substrate recognition by a S-adenosylhomocysteine hydrolase Lpg2021 from Legionella pneumophila.

S-Adenosyl-l-homocysteine hydrolase (SAHH) is a crucial enzyme that governs S-adenosyl methionine (SAM)-dependent methylation reactions within cells and regulates the intracellular concentration of SAH. Legionella pneumophila, the causative pathogen of Legionnaires' disease, encodes Lpg2021, which is the first identified dimeric SAHH in bacteria and is a promising target for drug development. Here, we report the structure of Lpg2021 in its ligand-free state and in complexes with adenine (ADE), adenosine (ADO), and 3-Deazaneplanocin A (DZNep). X-ray crystallography, isothermal titration calorimetry (ITC), and molecular docking were used to elucidate the binding mechanisms of Lpg2021 to its substrates and inhibitors. Virtual screening was performed to identify potential Lpg2021 inhibitors. This study contributes a novel perspective to the understanding of SAHH evolution and establishes a structural framework for designing specific inhibitors targeting pathogenic Legionella pneumophila SAHH.

Development of SPQC sensor based on the specific recognition of TAL-effectors for locus-specific detection of 6-methyladenine in DNA.

N6-methyladenosine (6mA) plays a pivotal role in diverse biological processes, including cancer, bacterial toxin secretion, and bacterial drug resistance. However, to date there has not been a selective, sensitive, and simple method for quantitative detection of 6mA at single base resolution. Herein, we present a series piezoelectric quartz crystal (SPQC) sensor based on the specific recognition of transcription-activator-like effectors (TALEs) for locus-specific detection of 6mA. Detection sensitivity is enhanced through the use of a hybridization chain reaction (HCR) in conjunction with silver staining. The limit of detection (LOD) of the sensor was 0.63 pM and can distinguish single base mismatches. We demonstrate the applicability of the sensor platform by quantitating 6mA DNA at a specific site in biological matrix. The SPQC sensor presented herein offers a promising platform for in-depth study of cancer, bacterial toxin secretion, and bacterial drug resistance.

Semiconservative transmission of DNA N 6-adenine methylation in a unicellular eukaryote.

Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

m6A RNA methylation: The latent string-puller in fibrosis.

Fibrosis is a pathological phenomenon characterized by the aberrant accumulation of extracellular matrix (ECM) in tissues. Fibrosis is a universally age-related disease involving that many organs and is the final stage of many chronic inflammatory diseases, which often threaten the patient's health. Undoubtedly, fibrosis has become a serious economic and health burden worldwide, However, the pathogenesis of fibrosis is complex. Further, the key molecules still remain to be unraveled. Hence, so far, there have been no effective treatments designed against the key targets of fibrosis. The methylation modification on the nitrogen atom at position 6 of adenine (m6A) is the most common mRNA modification in mammals. There is increasing evidence that m6A is actively involved in the pathogenesis of fibrosis. This review aims to highlight m6A-associated mechanisms and functions in several organic fibrosis, which implies that m6A is universal and critical for fibrosis and summarize the outlook of m6A in the treatment of fibrosis. This may light up the unknown aspects of this condition for researchers interested to explore fibrosis further.

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Base editing correction of OCRL in Lowe syndrome: ABE-mediated functional rescue in patient-derived fibroblasts.

Lowe syndrome, a rare X-linked multisystem disorder presenting with major abnormalities in the eyes, kidneys, and central nervous system, is caused by mutations in OCRL gene (NG_008638.1). Encoding an inositol polyphosphate 5-phosphatase, OCRL catalyzes the hydrolysis of PI(4,5)P2 into PI4P. There are no effective targeted treatments for Lowe syndrome. Here, we demonstrate a novel gene therapy for Lowe syndrome in patient fibroblasts using an adenine base editor (ABE) that can efficiently correct pathogenic point mutations. We show that ABE8e-NG-based correction of a disease-causing mutation in a Lowe patient-derived fibroblast line containing R844X mutation in OCRL gene, restores OCRL expression at mRNA and protein levels. It also restores cellular abnormalities that are hallmarks of OCRL dysfunction, including defects in ciliogenesis, microtubule anchoring, α-actinin distribution, and F-actin network. The study indicates that ABE-mediated gene therapy is a feasible treatment for Lowe syndrome, laying the foundation for therapeutic application of ABE in the currently incurable disease.