RNA Methylation m6A: A New Code and Drug Target?

The m⁶A‐RNA modification is a dynamic and reversible process, which has emerged as a new RNA code for the regulation of gene expression. The functional network of methyltransferases (writers), demethylases (erasers), and binding proteins (readers) modulate the level of m⁶A modification. Dysfunction of RNA methylation has been associated with various fundamental biological processes and human diseases. Herein, we briefly introduce an understanding‐enabled manipulation on m⁶A‐RNA modification with an emphasis on the use of small‐molecule intervention.      

Identification of Flavin Mononucleotide as a Cell‐Active Artificial N6‐Methyladenosine RNA Demethylase

N⁶‐Methyladenosine (m⁶A) 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 m⁶A residues in mRNA. Herein, the first identification of a small‐molecule modulator that functions as an artificial m⁶A demethylase is reported. Flavin mononucleotide (FMN), the metabolite produced by riboflavin kinase, mediates substantial photochemical demethylation of m⁶A residues of RNA in live cells. This study provides a new perspective to the understanding of demethylation of m⁶A residues in mRNA and sheds light on the development of powerful small molecules as RNA demethylases and new probes for use in RNA biology.     

Mapping N6-Methyladenosine (m6A) in RNA: Established Methods,Remaining Challenges,and Emerging Approaches

N⁶-Methyladenosine (m⁶A) is the most abundant internal modification in eukaryotic mRNA. Specific m⁶A reader and eraser proteins link this modification to many aspects of mRNA metabolism and regulate its levels in a dynamic way. Precise localization and quantification in varying biological samples is, therefore, relevant to understand the functional role of m⁶A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m⁶A mapping. Starting with the seminal m⁶A-sequencing techniques based on immunoprecipitation, we will highlight technical improvements by photo-cross-linking and remaining challenges. As an alternative, antibody-free approaches will be presented. These include wild-type or engineered m⁶A-sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m⁶A. Finally, single-molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.     

2-萘-(OCH2CH2)n-金刚胺与6-单取代环糊精衍生物的光诱导电子转移反应

A number of naphthalene donor compounds that possess an adamantanamine binding moiety and an (OCH2CH2)n (nn1, 2, 3, 4, 6, 8) spacer were synthesized. The fluorescence quenching between these donor substrates and mono-6-O-p-nitrobenzoyl-β-cyclodextrin (pNBCD) and mono-6-O-m-nitrobenzoyl-β-cyclodextrin(mNBCD) was studied in detail. It was found that very efficient fluorescence quenching could occur in these supramolecular systems. This quenching was attributed to the photoinduced electron transfer inside the supramolecular assembly between the naphthalene donors and cyclodextrin acceptors. Detailed Stern-Volmer constants were measured and they were partitioned into dynamic Stern-Volmer quenching constants and static binding constants. It was demonstrated that the binding constants between all the naphthalene compounds and cyclodextrins are the same as they possess the same binding site, i.e., adamantanamine.     

Synthesis and NMR characterization of 6‐Phenyl‐6‐deoxy‐2,3‐di‐O‐methylcellulose

Cellulose ( 1 ) was converted for the first time to 6‐phenyl‐6‐deoxy‐2,3‐di‐O‐methylcellulose ( 6 ) in 33% overall yield. Intermediates in the five‐step conversion of 1 to­ 6 were: 6‐O‐tritylcellulose ( 2 ), 6‐O‐trityl‐2,3‐di‐O‐methylcellulose ( 3 ), 2,3‐di‐O‐methylcellulose ( 4 ); and 6‐bromo‐6‐deoxy‐2,3‐di‐O‐methylcellulose ( 5 ). Elemental and quantitative carbon‐13 analyses were concurrently used to verify and confirm the degrees of substitution in each new polymer. Gel permeation chromotography (GPC) data were generated to monitor the changes in molecular weight (DP_w) as the synthesis progressed, and the compound average decrease in cellulose DP_w was ～ 27%. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to characterize the decomposition of all polymers. The degradation temperatures ( °C) and percent char at 500 °C of cellulose derivatives 2 to 6 were 308.6 and 6.3%, 227.6 °C and 9.7%, 273.9 °C and 30.2%, 200.4 °C and 25.6%, and 207.2 °C and 27.0%, respectively. The glass transition temperature (T_g) of­6‐O‐tritylcellulose by dynamic mechanical analysis (DMA) occurred at 126.7 °C and the modulus (E′, Pa) dropped 8.9 fold in the transition from ?150 °C to + 180 °C (6.6 × 10⁹ to 7.4 × 10⁸ Pa). Modulus at 20 °C was 3.26 × 10⁹ Pa. Complete proton and carbon‐13 chemical shift assignments of the repeating unit of the title polymer were made by a combination of the HMQC and COSY NMR methods. Ultimate non‐destructive proof of carbon–carbon bond formation at C6 of the anhydroglucose moiety was established by generating correlations between resonances of CH₂6 (anhydroglucose) and C1′, H2′, and H6′ of the attached aryl ring using the heteronuclear multiple‐bond correlation (HMBC) method. In this study, we achieved three major objectives: (a) new methodologies for the chemical modification of cellulose were developed; (b) new cellulose derivatives were designed, prepared and characterized; (c) unequivocal structural proof for carbon–carbon bond formation with cellulose was derived non‐destructively by use of one‐ and two‐dimensional NMR methods. Copyright © 2002 John Wiley & Sons, Ltd.     

Double‐Oxygen‐Atom Transfer in Reactions of CemO2m+ (m=2–6) with C2H2

Cerium oxide cluster cations (Ce_mO_n⁺, m=2–16; n=2m, 2m±1 and 2m±2) are prepared by laser ablation and reacted with acetylene (C₂H₂) in a fast‐flow reactor. A time‐of‐flight mass spectrometer is used to detect the cluster distribution before and after the reactions. Reactions of stoichiometric Ce_mO_2m⁺ (m=2–6) with C₂H₂ produce Ce_mO_2m?2⁺ clusters, which indicates a “double‐oxygen‐atom transfer” reaction Ce_mO_2m⁺+C₂H₂→Ce_mO_2m?2⁺+(CHO)₂ (ethanedial). A single‐oxygen‐atom transfer reaction channel is also identified as Ce_mO_2m⁺+C₂H₂→Ce_mO_2m?1⁺+C₂H₂O (at least for m=2 and 3). Density functional theory calculations are performed to study reaction mechanisms of Ce₂O₄⁺+C₂H₂, and the calculated results confirm that both the single‐ and double‐oxygen‐atom transfer channels are thermodynamically and kinetically favourable.     

Crystallographic report: {[Zn(O2CC6H4NO2‐m)(1,10‐phenanthroline)2] O2CC6H4NO2‐m}·2H2O·HO2CC6H4NO2‐m

The structure of {[Zn(O₂CC₆H₄NO₂‐m)(1,10‐phenanthroline)₂]O₂CC₆H₄NO₂‐m}·2H₂O·HO₂CC₆H₄NO₂‐m features chelating m‐nitrobenzoate and 1,10‐phenanthroline ligands so that a distorted octahedron N₄O₂ coordination geometry results. Copyright © 2005 John Wiley & Sons, Ltd.     

Preparation of a multi‐hollow magnetic molecularly imprinted polymer for the selective enrichment of indolebutyric acid

A simple strategy was developed for the preparation of multi‐hollow magnetic molecularly imprinted polymers by incorporating 3‐indolebutyric acid and ferroferric oxide nanoparticles simultaneously into a poly(styrene‐co‐methacrylic acid) copolymer matrix. The as prepared absorbents were characterized using scanning electron microscopy, Fourier‐transform infrared spectroscopy and mercury porosimetry. The adsorption isotherms of indolebutyric acid revealed that there are two types of affinity binding sites in the absorbents. The apparent maximum binding capacity and dissociation constant were 17.88 mg/g and 158.7 μg/mL for high‐affinity binding sites and 9.310 mg/g and 35.04 μg/mL for low‐affinity binding sites, respectively. The results testified that multi‐hollow magnetic molecularly imprinted polymers possessed excellent recognition capacity and fast kinetic binding behavior to the objective molecules due to the high specific surface area as large as 511.3 m²/g. Recoveries of 75.5–86.8% were obtained for the indolebutyric acid spiked at three concentration levels in blank and pear samples.     

Mannose‐Binding Geometry of Pradimicin A

Pradimicins (PRMs) and benanomicins are the only family of non‐peptidic natural products with lectin‐like properties, that is, they recognize D ‐mannopyranoside (Man) in the presence of Ca²⁺ ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti‐HIV activities through binding to Man‐containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate‐forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM‐A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM‐A and Man. Evaluation of intermolecular distances by solid‐state NMR spectroscopy revealed that the C2–C4 region of Man is in close contact with the primary binding site of PRM‐A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co‐precipitation experiments using deoxy‐Man derivatives, leading to the proposal that PRM‐A binds not only to terminal Man residues at the non‐reducing end of glycans, but also to internal 6‐substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM‐A.     

Membrane Composition Effects on the Analytical Behavior of Solid Contact Potentiometric Pb2+ Selective Electrodes Based on Diazadibenzo‐18‐crown‐6 Ionophores

The effect of component contents and membrane thickness on the detection limit (DL), slope (m), linear range (LR) and response time (RT) of Pb²⁺ solid contact potentiometric ion selective electrodes (SCISE) based on 4,10‐diaza‐2,3,11,12‐dibenzo‐18‐crown‐6 (1), 4,10‐diaza‐2,3,11,12‐di(4‐tert‐butylbenzo)‐18‐crown‐6 (2) and 4,10‐diaza‐2,3,11,12‐dibenzo‐18‐crown‐6‐N,N′‐di(carboxymethyl) (3) as ionophores was studied by open circuit potential (OCP) and electrochemical impedance spectroscopy (EIS) measurements. The use of an intermediate layer of poly(3‐octyl)thiophene between the gold substrate and the selective membrane was explored. SCISE prepared showing the best responses had typical DL, m, LR and RT values of 10^?6 M, 29 mV/dec, 10^?5 to 10^?3 M and 2 minutes.     

Layer‐by‐layer Assembled Multilayers of Graphene/Mono‐(6‐amino‐6‐deoxy)‐β‐cyclodextrin for Detection of Dopamine

Graphene/mono‐(6‐amino‐6‐deoxy)‐β‐cyclodextrin multilayer films composed of graphene sheet (GS) and mono‐(6‐amino‐6‐deoxy)‐β‐cyclodextrin (NH₂‐β‐CD) were fabricated easily by two steps. First, negatively charged graphene oxide (GO) and positively charged mono‐(6‐amino‐6‐deoxy)‐β‐cyclodextrin (NH₂‐β‐CD) were layer‐by‐layer (LBL) self‐assembled on glassy carbon electrode (GCE) modified with a layer of poly(diallyldimethylammonium chloride) (PDDA). Then graphene/mono‐(6‐amino‐6‐deoxy)‐β‐cyclodextrin (GS/NH₂‐β‐CD) multilayer films were built up by electrochemical reduction of graphene oxide/mono‐(6‐amino‐6‐deoxy)‐β‐cyclodextrin (GO/NH₂‐β‐CD). Combining the high surface area of GS and the active recognition sites on β‐cyclodextrin (β‐CD), the GS/NH₂‐β‐CD multilayer films show excellent electrochemical sensing performance for the detection of DA with an extraordinary broad linear range from 2.53 to 980.05 µmol·L^?1. This study offers a simple route to the controllable formation of graphene‐based electrochemical sensor for the detection of DA.     

A re‐examination of the sub‐Tm exotherm in polyamide 6: The roles of thermal history,water and clay

The sub‐T_m exotherms in polyamide 6 (PA6) have been carefully re‐examined by differential scanning calorimetry and X‐ray diffraction, considering the effects of processing and thermal history, addition of water and clay. The results obtained cast doubt on Khanna's proposal that sub‐T_m exotherm in PA6 comes from the release of strain energy absorbed during processing, and suggested that the origin of sub‐T_m exotherm is the γ?α transformation at the premelting temperature, namely, the less thermodynamically stable γ‐form (γ^ns) transforming into the more thermodynamically stable α‐form (α^s). The presence of water or clay in PA6 samples facilitated the formation of γ^ns at corresponding cooling rates, and enhanced the development of sub‐T_m exotherms. During the heating scan of PA6/clay composites, the initial γ^ns can be transformed into more stable (γ^s)^t and α^s at the same time, which can be thought as the origin of their sub‐T_m events. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 2385–2393, 2009     

A Molecular Placeholder Strategy To Access a Family of Transition‐Metal‐Functionalized Vanadium Oxide Clusters

Systematic access to metal‐functionalized polyoxometalates has thus far been limited to lacunary tungsten oxide and molybdenum oxide clusters. The first controlled, stepwise bottom‐up assembly route to metal‐functionalized molecular vanadium oxides is now presented. A di‐vacant vanadate cluster with two metal binding sites, (DMA)₂[V₁₂O₃₂Cl]^3? (DMA=dimethylammonium) is formed spontaneously in solution and characterized by single‐crystal X‐ray diffraction, ESI mass spectrometry, ⁵¹V NMR spectroscopy, and elemental analyses. In the cluster, the metal binding sites are selectively blocked by hydrogen‐bonded DMA placeholder cations. Reaction of the cluster with transition metals TM (Fe³⁺, Co²⁺, Cu²⁺, Zn²⁺) gives access to mono‐functionalized vanadate clusters (DMA)[{TM(L)}V₁₂O₃₂Cl]^n? (L=ligand). Metal binding is accomplished by significant distortions of the vanadium oxide framework reminiscent of a pincer movement. Cluster stability under technologically relevant conditions in the solid‐state and solution is demonstrated.     

Computational design and fabrication of core–shell magnetic molecularly imprinted polymer for dispersive micro‐solid‐phase extraction coupled with high‐performance liquid chromatography for the determination of rhodamine 6G

A novel core–shell magnetic nano‐adsorbent with surface molecularly imprinted polymer coating was fabricated and then applied to dispersive micro‐solid‐phase extraction followed by determination of rhodamine 6G using high‐performance liquid chromatography. The molecularly imprinted polymer coating was prepared by copolymerization of dopamine and m‐aminophenylboronic acid (functional monomers), in the presence of rhodamine 6G (template). The selection of the suitable functional monomers was based on the interaction between different monomers and the template using the density functional theory. The ratios of the monomers to template were further optimized by an OA₉ (3⁴) orthogonal array design. The binding performances of the adsorbent were evaluated by static, kinetic, and selective adsorption experiments. The results reveal that the adsorbent possesses remarkable affinity and binding specificity for rhodamine 6G because of the enhanced Lewis acid‐base interaction between the B(Ш) embedded in the imprinted cavities and the template. The nano‐adsorbent was successfully applied to dispersive micro‐solid‐phase extraction coupled to high‐performance liquid chromatography for the trace determination of rhodamine 6G in samples with a detection limit of 2.7 nmol/L. Spiked recoveries ranged from 93.0–99.1, 89.5–92.7, and 86.9–105% in river water, matrimony vine and paprika samples, respectively, with relative standard deviations of less than 4.3%.     

ACE applied to the quantitative characterization of benzo‐18‐crown‐6‐ether binding with alkali metal ions in a methanol–water solvent system

ACE was applied to the quantitative evaluation of noncovalent binding interactions between benzo‐18‐crown‐6‐ether (B18C6) and several alkali metal ions, Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺, in a mixed binary solvent system, methanol–water (50/50 v/v). The apparent binding (stability) constants (K_b) of B18C6–alkali metal ion complexes in the hydro‐organic medium above were determined from the dependence of the effective electrophoretic mobility of B18C6 on the concentration of alkali metal ions in the BGE using a nonlinear regression analysis. Before regression analysis, the mobilities measured by ACE at ambient temperature and variable ionic strength of the BGE were corrected by a new procedure to the reference temperature, 25°C, and the constant ionic strength, 10 mM . In the 50% v/v methanol–water solvent system, like in pure methanol, B18C6 formed the strongest complex with potassium ion (log K_b=2.89±0.17), the weakest complex with cesium ion (log K_b=2.04±0.20), and no complexation was observed between B18C6 and the lithium ion. In the mixed methanol–water solvent system, the binding constants of the complexes above were found to be about two orders lower than in methanol and about one order higher than in water.     

Influence of alkoxy tail length on the self‐organization of hairy‐rod polymers based on mesogen‐jacketed liquid crystalline polymers

A series of hairy‐rod polymers, poly{2,5‐bis[(4‐alkoxyphenyl)oxycarbonyl]styrenes} (P‐OCm, m = 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18) were designed and successfully synthesized via free radical polymerization. The chemical structure of the monomers was confirmed by elemental analysis, ¹H NMR and ¹³C NMR. The molecular characterizations of the polymers were performed with ¹H NMR and gel permeation chromatography. The phase structures and transitions of the polymers were investigated by the combination of techniques including differential scanning calorimetry, wide‐angle X‐ray diffraction, polarized optical microscopy, and rheological measurement. The experimental results revealed that the self‐assembly behaviors of P‐OCm changed with the increase in m. First, the P‐OCm (m = 1, 2) showed only a stable liquid crystalline phase above T_g. Second, with the increasing length of alkoxy tails, the P‐OCm (m = 4, 6, 8) presented a re‐entrant isotropic phase above T_g and a liquid crystalline phase at higher temperature. Third, the P‐OCm (m = 10, 12, 14, 16, 18) exhibited an unusual re‐entrant isotropic phase which was separating SmA (in low temperature) and columnar phases (in high temperature). It was the first time that mesogen‐jacketed liquid crystalline polymers formed smectic phase, re‐entrant isotropic phase, and columnar phases in one polymer due to the microphase separation and the driving force of the entropy. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

1, 9‐Dihydro‐purine‐6‐thione Derivatives of the d8–d10 Metal Ions (PdII,PtII, and CuI): Synthesis,Spectroscopy, and Structures

Reaction of 1, 9‐dihydro‐purine‐6‐thione (puSH₂) in presence of aqueous sodium hydroxide with PdCl₂(PPh₃)₂ suspended in ethanol formed [Pd(κ²‐N⁷,S‐puS)(PPh₃)₂] ( 1 ). Similarly, complexes [Pd(κ²‐N⁷,S‐puS)(κ²‐P, P‐L‐L)] ( 2 – 4 ) {L‐L = dppm (m = 1) ( 2 ), dppp (m = 3) ( 3 ), dppb (m = 4) ( 4 )} were prepared using precursors the [PdCl₂(L‐L)] {L‐L = Ph₂P–(CH₂)_m–PPh₂}. Reaction of puSH₂ suspended in benzene with platinic acid, H₂PtCl₆, in ethanol in the presence of triethylamine followed by the addition of PPh₃ yielded the complex [Pt(κ²‐N⁷,S‐puS)(PPh₃)₂] ( 5 ). Complexes [Pt(κ²‐N⁷,S‐puS)(κ²‐P, P‐L‐L)] ( 6 – 8 ) {L‐L = dppm ( 6 ), dppp ( 7 ), dppb ( 8 )} were prepared similarly. The 1, 9‐dihydro‐purine‐6‐thione acts as N⁷,S‐chelating dianion in compounds 1 – 8 . The reaction of copper(I) chloride [or copper(I) bromide] in acetonitrile with puSH₂ and the addition of PPh₃ in methanol yielded the same product, [Cu(κ²‐N⁷,S‐puSH)(PPh₃)₂] ( 9 ), in which the halogen atoms are removed by uninegative N, S‐chelating puSH^– anion. However, copper(I) iodide did not lose iodide and formed the tetrahedral complex, [CuI(κ¹‐S‐puSH₂)(PPh₃)₂] ( 10 ), in which the thio ligand is neutral. These complexes were characterized with the help of elemental analysis, NMR spectroscopy (¹H, ³¹P), and single‐crystal X‐ray crystallography ( 3 , 7 , 8 , 9 , and 10 ).