ZnO nanoparticles induce melanoma-like lesions via recruiting dermal dendritic cells in barrier-damaged skin in mice.

ZnO nanoparticles (NPs) are used in skin treatments and cosmetics, the toxicity of long-term and continuous exposure to ZnO NPs is unknown. Mice with epidermal barrier dysfunction revealed melanoma-like lesions after continuous exposure to ZnO NPs. However, the effects of metallic NPs on the skin microenvironment and immune system remain poorly understood. Mice with epidermal barrier failure were given continuous exposure to ZnO NPs for 7 weeks. The malignant transformation of melanocytes was induced with ZnO NPs 2.5 µg/ml for 72 h exposure. The supernatant of the culture medium from dendritic cells after being exposed to 10 µg/ml ZnO NPs for 24 h was applied to melanocytes to explore the effect of recruitment of DCs. The expressure of ZnO NPs resulted in a tendency of malignant transformation of melanocytes, the recruitment of DCs induces this process by produce inflammatory factors such as TNF-α. These DC-produced inflammatory factors, which were induced by ZnO NP exposure, increased the production of matrix metalloproteinases in melanocytes and expedited the malignant transformation process. Our findings revealed that the disrupted cutaneous microenvironment by ZnO NPs penetrated directly promoted the malignant transformation of melanocytes, which process also indirectly enhanced by the TNF-αsecreted from the recruited DCs.

Conformations of KRAS4B Affected by Its Partner Binding and G12C Mutation: Insights from GaMD Trajectory-Image Transformation-Based Deep Learning.

Binding of partners and mutations highly affects the conformational dynamics of KRAS4B, which is of significance for deeply understanding its function. Gaussian accelerated molecular dynamics (GaMD) simulations followed by deep learning (DL) and principal component analysis (PCA) were carried out to probe the effect of G12C and binding of three partners NF1, RAF1, and SOS1 on the conformation alterations of KRAS4B. DL reveals that G12C and binding of partners result in alterations in the contacts of key structure domains, such as the switch domains SW1 and SW2 together with the loops L4, L5, and P-loop. Binding of NF1, RAF1, and SOS1 constrains the structural fluctuation of SW1, SW2, L4, and L5; on the contrary, G12C leads to the instability of these four structure domains. The analyses of free energy landscapes (FELs) and PCA also show that binding of partners maintains the stability of the conformational states of KRAS4B while G12C induces greater mobility of the switch domains SW1 and SW2, which produces significant impacts on the interactions of GTP with SW1, L4, and L5. Our findings suggest that partner binding and G12C play important roles in the activity and allosteric regulation of KRAS4B, which may theoretically aid in further understanding the function of KRAS4B.

Aggregation-Induced Emission-Active Photosensitizer-Mediated Photodynamic Therapy for Anti-Psoriasis.

Hyperproliferative keratinocytes and subcutaneous inflammation contribute to the characteristic symptoms of psoriasis, including erythema, scales, or scaly plaques on the skin. These symptoms significantly affect patients' quality of life and cause severe physical and psychological distress. However, current treatment strategies have limited therapeutic effect and may lead to adverse side effects. In this study, we present the novel organic photosensitizer TBTDC [5-(((5-(7-(4-(diphenylamino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)thiophen-2-yl)methylene)amino)-3-methylthiophene-2,4-dicarbonitrile] nanoparticles (NPs) with aggregation-induced emission (AIE) characteristics to mediate photodynamic therapy (TBTDC NP-PDT) for psoriasis treatment. We demonstrate that TBTDC NPs effectively generate reactive oxygen species upon light irradiation and lead to significant apoptosis of psoriatic keratinocytes. Furthermore, TBTDC NPs exhibit high cellular uptake in diseased keratinocytes and induce endoplasmic reticulum stress (ERS)-mediated autophagy, which can also enhance apoptosis. Importantly, TBTDC NPs show no cytotoxicity toward keratinocytes. These unique properties of TBTDC NPs enable remarkable therapeutic effects against psoriasis-like skin lesions and related inflammation in vivo. Overall, our AIE-active TBTDC NP-PDT represents a promising strategy for treating psoriasis in clinical settings.

Sulfur-based fluorescent probes for biological analysis: A review.

The widespread adoption of small-molecule fluorescence detection methodologies in scientific research and industrial contexts can be ascribed to their inherent merits, including elevated sensitivity, exceptional selectivity, real-time detection capabilities, and non-destructive characteristics. In recent years, there has been a growing focus on small-molecule fluorescent probes engineered with sulfur elements, aiming to detect a diverse array of biologically active species. This review presents a comprehensive survey of sulfur-based fluorescent probes published from 2017 to 2023. The diverse repertoire of recognition sites, including but not limited to N, N-dimethylthiocarbamyl, disulfides, thioether, sulfonyls and sulfoxides, thiourea, thioester, thioacetal and thioketal, sulfhydryl, phenothiazine, thioamide, and others, inherent in these sulfur-based probes markedly amplifies their capacity for detecting a broad spectrum of analytes, such as metal ions, reactive oxygen species, reactive sulfur species, reactive nitrogen species, proteins, and beyond. Owing to the individual disparities in the molecular structures of the probes, analogous recognition units may be employed to discern diverse substrates. Subsequent to this classification, the review provides a concise summary and introduction to the design and biological applications of these probe molecules. Lastly, drawing upon a synthesis of published works, the review engages in a discussion regarding the merits and drawbacks of these fluorescent probes, offering guidance for future endeavors.

The impact of PET/CT and brain MRI for metastasis detection among patients with clinical T1-category lung cancer: Findings from a large-scale cohort study.

PURPOSE: [18F]-FDG PET/CT and brain MRI are common approaches to detect metastasis in patients of lung cancer. Current guidelines for the use of PET/CT and MRI in clinical T1-category lung cancer lack risk-based stratification and require optimization. This study stratified patients based on metastatic risk in terms of the lesions' size and morphological characteristics. METHODS: The detection rate of metastasis was measured in different sizes and morphological characteristics (solid and sub-solid) of tumors. To confirm the cut-off value for discriminating metastasis and overall survival (OS) prediction, the receiver operating characteristic (ROC) analysis was performed based on PET/CT metabolic parameters (SUVmax/SUVmean/SULpeak/MTV/TLG), followed by Kaplan-Meier analysis for survival in post-operation patients with and without PET/CT plus MRI. RESULTS: 2,298 patients were included. No metastasis was observed in patients with solid nodules < 8.0 mm and sub-solid nodules < 10.0 mm. The cut-off of PET/CT metabolic parameters on discriminating metastasis were 1.09 (SUVmax), 0.26 (SUVmean), 0.31 (SULpeak), 0.55 (MTV), and 0.81 (TLG), respectively. Patients undergoing PET/CT plus MRI exhibited longer OS compared to those who did not receive it in solid nodules ≥ 8.0 mm & sub-solid nodules ≥ 10.0 mm (HR, 0.44; p < 0.001); in solid nodules ≥ 8.0 mm (HR, 0.12; p<0.001) and in sub-solid nodules ≥ 10.0 mm (HR; 0.61; p=0.075), respectively. Compared to patients with metabolic parameters lower than cut-off values, patients with higher metabolic parameters displayed shorter OS: SUVmax (HR, 12.94; p < 0.001), SUVmean (HR, 11.33; p <0.001), SULpeak (HR, 9.65; p < 0.001), MTV (HR, 9.16; p = 0.031), and TLG (HR, 12.06; p < 0.001). CONCLUSION: The necessity of PET/CT and MRI should be cautiously evaluated in patients with solid nodules < 8.0 mm and sub-solid nodules < 10.0 mm, however, these examinations remained essential and beneficial for patients with solid nodules ≥ 8.0 mm and sub-solid nodules ≥ 10.0 mm.

Molecular Mechanism of Phosphorylation-Mediated Impacts on the Conformation Dynamics of GTP-Bound KRAS Probed by GaMD Trajectory-Based Deep Learning.

The phosphorylation of different sites produces a significant effect on the conformational dynamics of KRAS. Gaussian accelerated molecular dynamics (GaMD) simulations were combined with deep learning (DL) to explore the molecular mechanism of the phosphorylation-mediated effect on conformational dynamics of the GTP-bound KRAS. The DL finds that the switch domains are involved in obvious differences in conformation contacts and suggests that the switch domains play a key role in the function of KRAS. The analyses of free energy landscapes (FELs) reveal that the phosphorylation of pY32, pY64, and pY137 leads to more disordered states of the switch domains than the wild-type (WT) KRAS and induces conformational transformations between the closed and open states. The results from principal component analysis (PCA) indicate that principal motions PC1 and PC2 are responsible for the closed and open states of the phosphorylated KRAS. Interaction networks were analyzed and the results verify that the phosphorylation alters interactions of GTP and magnesium ion Mg2+ with the switch domains. It is concluded that the phosphorylation pY32, pY64, and pY137 tune the activity of KRAS through changing conformational dynamics and interactions of the switch domains. We anticipated that this work could provide theoretical aids for deeply understanding the function of KRAS.

Mutations influence the conformational dynamics of the GDP/KRAS complex.

Mutations near allosteric sites can have a significant impact on the function of KRAS. Three specific mutations, K104Q, G12D/K104Q, and G12D/G75A, which are located near allosteric positions, were selected to investigate the molecular mechanisms behind mutation-induced influences on the activity of KRAS. Gaussian accelerated molecular dynamics (GaMD) simulations followed by the principal component analysis (PCA) were performed to improve the sampling of conformational states. The results revealed that these mutations significantly alter the structural flexibility, correlated motions, and dynamic behavior of the switch regions that are essential for KRAS binding to effectors or regulators. Furthermore, the mutations have a significant impact on the hydrogen bonding interactions between GDP and the switch regions, as well as on the electrostatic interactions of magnesium ions (Mg2+) with these regions. Our results verified that these mutations strongly influence the binding of KRAS to its effectors or regulators and allosterically regulate the activity. We believe that this work can provide valuable theoretical insights into a deeper understanding of KRAS function.Communicated by Ramaswamy H. Sarma.

Conformational States of the GDP- and GTP-Bound HRAS Affected by A59E and K117R: An Exploration from Gaussian Accelerated Molecular Dynamics.

The HRAS protein is considered a critical target for drug development in cancers. It is vital for effective drug development to understand the effects of mutations on the binding of GTP and GDP to HRAS. We conducted Gaussian accelerated molecular dynamics (GaMD) simulations and free energy landscape (FEL) calculations to investigate the impacts of two mutations (A59E and K117R) on GTP and GDP binding and the conformational states of the switch domain. Our findings demonstrate that these mutations not only modify the flexibility of the switch domains, but also affect the correlated motions of these domains. Furthermore, the mutations significantly disrupt the dynamic behavior of the switch domains, leading to a conformational change in HRAS. Additionally, these mutations significantly impact the switch domain's interactions, including their hydrogen bonding with ligands and electrostatic interactions with magnesium ions. Since the switch domains are crucial for the binding of HRAS to effectors, any alterations in their interactions or conformational states will undoubtedly disrupt the activity of HRAS. This research provides valuable information for the design of drugs targeting HRAS.

EVLncRNAs 3.0: an updated comprehensive database for manually curated functional long non-coding RNAs validated by low-throughput experiments.

Long noncoding RNAs (lncRNAs) have emerged as crucial regulators across diverse biological processes and diseases. While high-throughput sequencing has enabled lncRNA discovery, functional characterization remains limited. The EVLncRNAs database is the first and exclusive repository for all experimentally validated functional lncRNAs from various species. After previous releases in 2018 and 2021, this update marks a major expansion through exhaustive manual curation of nearly 25 000 publications from 15 May 2020, to 15 May 2023. It incorporates substantial growth across all categories: a 154% increase in functional lncRNAs, 160% in associated diseases, 186% in lncRNA-disease associations, 235% in interactions, 138% in structures, 234% in circular RNAs, 235% in resistant lncRNAs and 4724% in exosomal lncRNAs. More importantly, it incorporated additional information include functional classifications, detailed interaction pathways, homologous lncRNAs, lncRNA locations, COVID-19, phase-separation and organoid-related lncRNAs. The web interface was substantially improved for browsing, visualization, and searching. ChatGPT was tested for information extraction and functional overview with its limitation noted. EVLncRNAs 3.0 represents the most extensive curated resource of experimentally validated functional lncRNAs and will serve as an indispensable platform for unravelling emerging lncRNA functions. The updated database is freely available at https://www.sdklab-biophysics-dzu.net/EVLncRNAs3/.

Recurrence of solitary fibrous tumor of the pleura with hypoglycemia (Doege-Potter Syndrome): a case report description.

Hypoglycemia has multiple causes, but the most common is a complication of insulin treatment. In addition to insulin therapy, tumors such as insulinomas of pancreatic origin and extrapancreatic tumors causing paraneoplastic syndromes should also be considered. Solitary fibrous tumors of the pleura (SFTP) is rare tumor, which when associated with hypoglycemia causes Doege-Potter syndrome. This article reports a case of a 69-year-old man with Doege-Potter syndrome and underwent the first surgical resection for SFTP. However, the tumor recurred 9 years later with hypoglycemic symptoms and implant metastasis. This recurrent tumor originated from the visceral pleura, was more aggressive and invaded the diaphragm and parietal pleura. After the second surgical removal of the tumor, the hypoglycemic symptoms disappeared.

Corilagin alleviates LPS-induced sepsis through inhibiting pyroptosis via targeting TIR domain of MyD88 and binding CARD of ASC in macrophages.

Sepsis is a dysregulated systemic inflammatory response caused by infection that leads to multiple organ injury and high mortality without effective treatment. Corilagin, a natural polyphenol extracted from traditional Chinese herbs, exhibits strong anti-inflammatory properties. However, the role for Corilagin in lipopolysaccharide (LPS)-induced sepsis and the molecular mechanisms underlying this process have not been completely explored. Here we determine the effect of Corilagin on LPS-treated mice and use a screening approach integrating surface plasmon resonance with liquid chromatography-tandem mass spectrometry (SPR-LC-MS/MS) to further explore the therapeutic target of Corilagin. We discovered that Corilagin significantly prolonged the survival time of septic mice, attenuated the multi-organ injury and the expression of pyroptosis-related proteins in tissues of LPS-treated mice. In vitro studies revealed that Corilagin inhibited pyroptosis and NLRP3 inflammasome activation in LPS-treated macrophages followed with ATP stimulation, as reflected by decreased levels of GSDMD-NT and activated caspase-1, and reduced ASC specks formation. Mechanistically, Corilagin alleviated the formation of ASC specks and blocked the interaction of ASC and pro-caspase1 by competitively binding with the caspase recruitment domain (CARD) of ASC. Additionally, Corilagin interrupted the TLR4-MyD88 interaction through targeting TIR domain of MyD88, leading to the inhibition of NF-κB activation and NLRP3 production. In addition, Corilagin downregulated genes associated with several inflammatory responses and inflammasome-related signaling pathways in LPS-stimulated macrophages. Overall, our results indicate that the inhibitory effect of Corilagin on pyroptosis through targeting TIR domain of MyD88 and binding the CARD domain of ASC in macrophages plays an essential role in protection against LPS-induced sepsis.

Dynamical characterization and multiple unbinding paths of two PreQ1 ligands in one pocket.

Riboswitches naturally regulate gene expression in bacteria by binding to specific small molecules. Class 1 preQ1 riboswitch aptamer is an important model not only for RNA folding but also as a target for designing small molecule antibiotics due to its well-known minimal aptamer domain. Here, we ran a total of 62.4 µs conventional and enhanced-sampling molecular dynamics (MD) simulations to characterize the determinants underlying the binding of the preQ1-II riboswitch aptamer to two preQ1 ligands in one binding pocket. Decomposition of binding free energy suggested that preQ1 ligands at α and ß sites interact with four nucleotides (G5, C17, C18, and A30) and two nucleotides (A12 and C31), respectively. Mg2+ ions play a crucial role in both stabilizing the binding pocket and facilitating ligand binding. The flexible preQ1 ligand at the ß site leads to the top of the binding pocket loosening and thus pre-organizes the riboswitch for ligand entry. Enhanced sampling simulations further revealed that the preQ1 ligand at the α site unbinds through two orthogonal pathways, which are dependent on whether or not a ß site preQ1 ligand is present. One of the two preQ1 ligands has been identified in the binding pocket, which will aid to identify the second preQ1 Ligand. Our work provides new information for designing robust ligands.

Genome-Wide Identification and Functional Characterization of FAR1-RELATED SEQUENCE (FRS) Family Members in Potato (Solanum tuberosum).

FAR1-RELATED SEQUENCE (FRS) transcription factors are generated by transposases and play vital roles in plant growth and development, light signaling transduction, phytohormone response, and stress resistance. FRSs have been described in various plant species. However, FRS family members and their functions remain poorly understood in vegetative crops such as potato (Solanum tuberosum, St). In the present study, 20 putative StFRS proteins were identified in potato via genome-wide analysis. They were non-randomly localized to eight chromosomes and phylogenetic analysis classified them into six subgroups along with FRS proteins from Arabidopsis and tomato. Conserved protein motif, protein domain, and gene structure analyses supported the evolutionary relationships among the FRS proteins. Analysis of the cis-acting elements in the promoters and the expression profiles of StFRSs in various plant tissues and under different stress treatments revealed the spatiotemporal expression patterns and the potential roles of StFRSs in phytohormonal and stress responses. StFRSs were differentially expressed in the cultivar "Xisen 6", which is exposed to a variety of stresses. Hence, these genes may be critical in regulating abiotic stress. Elucidating the StFRS functions will lay theoretical and empirical foundations for the molecular breeding of potato varieties with high light use efficiency and stress resistance.

Magnesium ions mediate ligand binding and conformational transition of the SAM/SAH riboswitch.

The SAM/SAH riboswitch binds S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) with similar affinities. Mg2+ is generally known to stabilize RNA structures by neutralizing phosphates, but how it contributes to ligand binding and conformational transition is understudied. Here, extensive molecular dynamics simulations (totaling 120 µs) predicted over 10 inner-shell Mg2+ ions in the SAM/SAH riboswitch. Six of them line the two sides of a groove to widen it and thereby pre-organize the riboswitch for ligand entry. They also form outer-shell coordination with the ligands and stabilize an RNA-ligand hydrogen bond, which effectively diminishes the selectivity between SAM and SAH. One Mg2+ ion unique to the apo form maintains the Shine-Dalgarno sequence in an autonomous mode and thereby facilitates its release for ribosome binding. Mg2+ thus plays vital roles in SAM/SAH riboswitch function.

Hemin as a General Static Dark Quencher for Constructing Heme Oxygenase-1 Fluorescent Probes.

The development of small-molecule probes suitable for live-cell applications remains challenging yet highly desirable. We report the first fluorescent probe, RBH, for imaging the heme oxygenase-1 (HO-1) activity in live cells after discovering hemin as a universal dark quencher. Hemin works via a static quenching mechanism and shows high quenching efficiency (>97 %) with fluorophores across a broad spectrum (λex =400-700 nm). The favorable properties of RBH (e.g. long excitation/emission wavelengths, fast response rate and high magnitude of signal increase) enable its use for determining HO-1 activity in complex biological samples. As HO-1 is involved in regulating antioxidant defence, iron homeostasis and gasotransmitter carbon monoxide production, we expect RBH to be a powerful tool for dissecting its functions. Also, the discovery of hemin as a general static dark quencher provides a straightforward strategy for constructing novel fluorescent probes for diverse biological species.

Magnesium ions mediate ligand binding and conformational transition of the SAM/SAH riboswitch.

The SAM/SAH riboswitch binds S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) with similar affinities. Mg 2+ is generally known to stabilize RNA structures by neutralizing phosphates, but how it contributes to ligand binding and conformational transition is understudied. Here, extensive molecular dynamics simulations (totaling 120 µs) identified over 10 inner-shell Mg 2+ ions in the SAM/SAH riboswitch. Six of them line the two sides of a groove to widen it and thereby pre-organize the riboswitch for ligand entry. They also form outer-shell coordination with the ligands and stabilize an RNA-ligand hydrogen bond, which effectively diminish the selectivity between SAM and SAH. One Mg 2+ ion unique to the apo form maintains the Shine-Dalgarno sequence in an autonomous mode and thereby facilitates its release for ribosome binding. Mg 2+ thus plays vital roles in SAM/SAH riboswitch function.

Electrochemiluminescence Sensor Based on CTS-MoS2 and AB@CTS with Functionalized Luminol for Detection of Malathion Pesticide Residues.

The accumulation of pesticide residues poses a significant threat to the health of people and the surrounding ecological systems. However, traditional methods are not only costly but require expertise in analysis. An electrochemiluminescence (ECL) aptasensor was developed using chitosan and molybdenum disulfide (CTS-MoS2), along with acetylene black (AB@CTS) for the rapid detection of malathion residues. Due to the weak interaction force, simple composite may lead to uneven dispersion; MoS2 and AB were dissolved in CTS solution, respectively, and utilized the biocompatibility of CTS to interact with each other on the electrode. The MoS2 nanosheets provided a large specific surface area, enhancing the utilization rate of catalytic materials, while AB exhibited excellent conductivity. Additionally, the dendritic polylysine (PLL) contained numerous amino groups to load abundant luminol to catalyze hydrogen peroxide (H2O2) and generate reactive oxygen species (ROS). The proposed ECL aptasensor obtained a low detection limit of 2.75 × 10-3 ng/mL (S/N = 3) with a good detection range from 1.0 × 10-2 ng/mL to 1.0 × 103 ng/mL, demonstrating excellent specificity, repeatability, and stability. Moreover, the ECL aptasensor was successfully applied for detecting malathion pesticide residues in authentic samples with recovery rates ranging from 94.21% to 99.63% (RSD < 2.52%). This work offers valuable insights for advancing ECL sensor technology in future applications.

Transcriptomic insights into the molecular mechanism for response of wild emmer wheat to stripe rust fungus.

Introduction: Continuous identification and application of novel resistance genes against stripe rust are of great importance for wheat breeding. Wild emmer wheat, Triticum dicoccoides, has adapted to a broad range of environments and is a valuable genetic resource that harbors important beneficial traits, including resistance to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). However, there has been a lack of systematic exploration of genes against Pst races in wild emmer wheat. Methods: Genome-wide transcriptome profiles were conducted on two wild emmer wheat genotypes with different levels of resistance to (Pst (DR3 exhibiting moderate (Pst resistance, and D7 displaying high (Pst resistance). qRT-PCR was performed to verify findings by RNA-seq. Results: A higher number of DEGs were identified in the moderately (Pst-resistant genotype, while the highly (Pst-resistant genotype exhibited a greater enrichment of pathways. Nonetheless, there were consistent patterns in the enrichment of pathways between the two genotypes at the same time of inoculation. At 24 hpi, a majority of pathways such as the biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, phenylalanine metabolism, and alpha-Linolenic acid metabolism exhibited significant enrichment in both genotypes. At 72 hpi, the biosynthesis of secondary metabolites and circadian rhythm-plant pathways were notably and consistently enriched in both genotypes. The majority of (WRKY, MADs , and AP2-ERF families were found to be involved in the initial stage of response to Pst invasion (24 hpi), while the MYB, NAC, TCP, and b-ZIP families played a role in defense during the later stage of Pst infection (72 hpi). Discussion: In this present study, we identified numerous crucial genes, transcription factors, and pathways associated with the response and regulation of wild emmer wheat to Pst infection. Our findings offer valuable information for understanding the function of crucial Pst-responsive genes, and will deepen the understanding of the complex resistance mechanisms against Pst in wheat.

Decoding the Identification Mechanism of an SAM-III Riboswitch on Ligands through Multiple Independent Gaussian-Accelerated Molecular Dynamics Simulations.

S-Adenosyl-l-methionine (SAM)-responsive riboswitches play a central role in the regulation of bacterial gene expression at the level of transcription attenuation or translation inhibition. In this study, multiple independent Gaussian-accelerated molecular dynamics simulations were performed to decipher the identification mechanisms of SAM-III (SMK) on ligands SAM, SAH, and EEM. The results reveal that ligand binding highly affects the structural flexibility, internal dynamics, and conformational changes of SAM-III. The dynamic analysis shows that helices P3 and P4 as well as two junctions J23 and J24 of SAM-III are highly susceptible to ligand binding. Analyses of free energy landscapes suggest that ligand binding induces different free energy profiles of SAM-III, which leads to the difference in identification sites of SAM-III on ligands. The information on ligand-nucleotide interactions not only uncovers that the π-π, cation-π, and hydrogen bonding interactions drive identification of SAM-III on the three ligands but also reveals that different electrostatic properties of SAM, SAH, and EEM alter the active sites of SAM-III. Meanwhile, the results also verify that the adenine group of SAM, SAH, and EEM is well recognized by conserved nucleotides G7, A29, U37, A38, and G48. We expect that this study can provide useful information for understanding the applications of SAM-III in chemical, synthetic RNA biology, and biomedical fields.