Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Advances in peptidyl Asx-specific ligases for the application of cyclic peptides / 药学学报

Asparaginyl endopeptidases (AEPs) in plants belong to the family of cysteine protease that undergo self-activation in the form of zymogen in acidic vacuole and play important physiological roles in maturation of seed storage proteins, protein degradation, programmed cell death and host defense. Bioprocessing enzymes (peptidyl Asx-specific ligases, PALs) that promote the maturation of cyclotides have recently been isolated and identified from several cyclotide-rich plants. PALs derived from AEPs can site-specifically catalyze the formation of asparagine or aspartate peptide bonds. Due to the advantages of relatively traceless peptide bonds and broad substrate spectrum and high catalytic efficiency, they have been playing important roles in the cyclization and modification of peptides and proteins, and are powerful tools for improving the stability of peptide drugs. This review describes the physiological functions of AEPs in plants and summarizes the discoveries, structural characteristics, catalytic mechanism and protein engineering of PALs, as well as the limitation of their applications and future trends. In addition, the applications of PALs in cyclotides biosynthesis and the development of macrocyclic peptides are highlighted, with the aim of providing a new idea for the biocatalytic synthesis of cyclic peptides.

Research progress of small molecule fluorescent probes for ferrous ion and heme / 药学学报

Small molecule fluorescent probes have gained widespread attention for their advantages of high selectivity, sensitivity, and easy to operate, and have played a critical role in the detection of various species. They have also demonstrated great potential in the field of biomedical research. Iron, as the most abundant transition metal in the human body, plays a vital role in many physiological functions. Due to the influence of the reductive microenvironment of cell, ferrous ion (Fe2+) is the main component of labile iron in living cells. Heme, consisting of Fe2+ and protoporphyrin IX, is one of the main signaling molecules that wrap biological iron in the human body, and also participates in many physiological and pathological processes. Therefore, the development of small molecule fluorescent probes for detecting Fe2+ and heme as effective monitoring tools will help to further understand their pathological and physiological functions, with potential applications in other fields. This review summarizes the research progress of small molecule fluorescent probes for Fe2+ and heme detection in recent years, and provides insights into future directions for their development.

Construction and in vitro evaluation of AIE self-assembled probe based on GSH response covalent cyclization / 国际生物医学工程杂志

Objective:To construct an aggregation induced emission (AIE) self-assembled probe based on glutathione (GSH) response covalent cyclization and evaluate it in vitro.Methods:The peptide sequence containing the 2-cyano-6-aminobenzothiazole-cysteine (CBT-Cys) condensation sequence was synthesized by the solid-phase peptide synthesis method. After coupling with an AIE molecule by click chemical reaction, an AIE self-assembled probe 1 based on GSH response covalent cyclization was constructed, and probe 2 lacking Cys structure was used as the control. The absorption and emission spectra of probes were tested and the specificity of probes to GSH was analyzed. The hydrodynamic diameter and structure of the probes after response were compared. The effects of different pH values, temperatures, probe concentrations, and GSH concentrations on fluorescence intensity were investigated. The toxicity of probes to tumor cells such as HeLa, HepG2 and MDA-MB-231 was evaluated.Results:After GSH response, the fluorescence of probe 1 was enhanced by about 6 times and that of probe 2 was enhanced by about 2 times; probe 1 was converted into a dimer with a hydrodynamic diameter of about 896.1 nm. Probe 2 lacked a cyclization motif and was converted into a monomer with a hydrodynamic diameter of about 427.4 nm. The fluorescence intensity of probe 1 was significantly higher than that of probe 2 at pH=7.0 and 37 ℃, and the toxicity of probes to tumor cells (HeLa, HepG2 and MDA-MB-231) was low.Conclusions:After the disulfide bond of probe 1 was reduced by GSH, the probe molecule lost the hydrophilic sequence, resulting in fluorescence turn-on (the first aggregation), and probe 1 immediately generates an AIE dimer (the second aggregation) because it contains a CBT-Cys cyclization sequence, which realizes the dual AIE effect compared with the single aggregation of probe 2, and significantly enhances the fluorescence emission. Probe 1 has better applicability in physiological environments, which provides an idea for in-situ generation of covalent cycling probes in vivo and is expected to be used in tumor imaging and treatment in the later stages.

Cyclization of α-conotoxin A10LPnIA with lysine assisted cyanuric chloride linker / 药学学报

The cyanuric chloride linkers have been used for cyclizing polypeptide, but not used for α-conotoxin, the peptides with rich disulfide bonds and more amino acid residues. In this study, cyclic peptides c[A10L]PnIA-1-4 were synthesized efficiently by lysine assisted cyanuric chloride linkers with 28.92%-52.00% yields. The activity evaluation showed that the IC50 values of c[A10L]PnIA-1 against α7 and α3β2 nAChR subtypes were 5 and 7 times higher than [A10L]PnIA respectively, and the subtype selectivity was maintained. The results of circular dichroism show that this cyclization method had no significant effect on its secondary structure. Compared with the commonly used head-to-tail cyclization in conotoxin cyclization, this method has the advantages of rapid reaction and high yield, which is expected to be further applied to the cyclization study of various α-conotoxins.

Research advances in methods of cyclezation mechanism of sesquiterpenes / 中国中药杂志

Terpenes are the largest group of natural products and contain the widest assortment of structural types. Terpene cyclization is also the most complex reaction found in nature. For a long time, terpenoids with diverse structures have attracted natural product chemists to explore their biosynthesis mechanism. Such a large number of terpene skeletons are catalyzed by enzymes called terpene synthase. Sesquiterpene synthase is a kind of terpene synthase, which can catalyze the cyclization of linear precursor farnesyl pyrophosphate(FPP) to sesquiterpene skeletons. Sesquiterpene synthase cyclize a single precursor FPP into many sesquiterpene skeletons. With the continuous discovery of sesquiterpene synthase, the cyclization mechanism of sesquiterpene synthase has been studied deeply. In recent years, with the development and improvement of isotope labeling of substrate FPP and structural analysis of sesquiterpene synthase, the structure and cyclization mechanism of sesquiterpene synthase have been studied more systematically and accurately. In this review, we reviewed the progress of the research methods on the mechanism of sesquiterpene cyclization by substrate isotope labeling and protein structure, as well as the summary and prospect of sesquiterpene synthase research.

The Cre-loxP system and its derivative systems: methodology research and applications in neuroscience / 药学学报

In scientific research, it is often needed to knock in, knock out, knock down, or overexpress a specific gene in model organisms or specific types of cells to achieve precise regulation of experimental independent variables. In this case, various transgenic mice are required. The cyclization recombinase (Cre) can directly interact with different loxP (locus X over P1) DNA sequences without any cofactors to perform specific gene knock-in or knock-out at specific targets. Because of its advantages of simple action principles, high spatial specificity, and high reorganization efficiency, the Cre-loxP system is widely used in scientific research. Furthermore, the CreERT2 system (mutant of the fusion protein of Cre and estrogen receptor ligand binding domain) and the tetracycline (Tet)-on/off system, derived from the Cre-loxP system, have made the recombination of the target gene occur in temporal-specificity on the basis of spatial-specificity. This dual specificity of time and space is indispensable for research in specific directions such as fear memory and engram cells on the basis of reducing the impacts on experimental animals. Therefore, these derived systems have broad application prospects.

Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization / 生物工程学报

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.

Advances in isopeptide bond-mediated molecular superglue / 生物工程学报

Isopeptide bond-mediated molecular superglue is the irreversible covalent bond spontaneously formed by the side chains of lysine (Lys) and asparagine/aspartic acid (Asn/Asp) residues. The peptide-peptide interaction is specific, stable, and can be achieved quickly without any particular physicochemical factor. In the light of recent progress by domestic and foreign researchers, here we summarize the origin, assembly system and mechanism of isopeptide bond reaction, as well as the molecular cyclization and protein topological structure mediated by it. The prospect for its application in synthetic vaccine, hydrogel and bacterial nanobiological reactor is further discussed.

Total synthesis of sauropunol A-D isolated from Sauropus rostratus genus plants / 中国药科大学学报

Sauropunol A-D potentially with anti-inflammatory,anti-bacterial activities were recently isolated from traditional Chinese medicinal plant Sauropus rostratus.Herein,we report the total synthesis of sauropunol (A-D) starting from a commercially available 2-deoxy-D-arabino-hexopyranose.The trifluoromethane sulfonation of intermediates could simultaneously trigger intramolecular cyclization to afford 3,6-anhydro hexofuranoside scaffold.The following deprotection reaction could produce sauropunol A,B and C/D with total yields of 21％,5％,and 17％ (isomer sauropunol C/D),respectively.Structures of the target compounds were confirmed by comparison with NMR spectroscopic properties of those previously reported.

Reviews on flavanone compounds synthesis research / 药学实践杂志

Research progress of flavanone compounds synthesis was reviewed ,various types of synthesis methods of fla‐vanone compounds were summarized ,and methodological support for flavanone compounds in new drug research and develop‐ment was provided .Research progress in synthesis of flavanone compounds includes chalcone cyclization ,Friedel‐Crafts reac‐tion ,Knoevenagel condensation ,Hoesch single acetoxylation ,and asymmetric synthesis of flavanone compounds .Solvent‐free synthesis of flavanone compounds and other green chemistry methods were also introduced .

Synthesis and microbial screening of 6-pyridin-4-yl-2, 3-dihydro-1Hpyridazin-pyridazin-4-one derivatves.

6-pyridin-4-yl-2, 3-dihydro-1H-pyridazin-4-one derivatives were synthesized by a simple method. The isonicotinic acid hydrazide was made to react with acetaldehyde and acetone. This results into the formation of the respective hydrazones. These hydrazones formed were cyclized to the pyridazine derivatives. All synthesized compounds were subjected to antimicrobial activity.

Green approach for the synthesis of silver-nanoparticles and efficient use in oxidative cyclization.

Highly concentrated and stable suspension of Ag-nanoparticles were synthesized by the chemical reduction of AgNO3 in a reductant using a macrocyclic Schiff base ligand benzildiethylenetriamine and low molecular weight organic compound, thiosalicylic acid(TSA) used as a protective agent. The average size of the silver nanoparticles prepared from this method was less than 20 nm, which allowed low-temperature sintering of the metal. Ag-nanoparticles provide an efficient catalysis for the synthesis of heterocyclic compounds by coupling salicyldehyde with aliphatic amines through the oxidative cyclization of Schiff base using 10 mol. % of Ag-nanoparticles in the presence of Na2CO3 in MeOH. This method avoids the usage of metal oxides or organic oxidizing agents.

N-Terminal Cyclization of Peptides in LargE-scale Protein Identification Based on Biological Mass Spectrometry / 分析化学

Biological mass spectrometry has been developed for the largE-scale protein identification. The successful identification of protein in proteomic study is based on an effective match of MS data to the sequence in database. Because of the diversity and heterogeneity of protein modification, the experimental data obtained by mass spectrometry does not match the theoretical value sometimes, which makes about 90 percent or more of the tandem mass spectra not be effectively identified. This has become one of the most important technique issues to be resolved in current proteome research. The N-terminal cyclization of peptides, as one of a variety of modification introduced in sample preparation, has been preliminarily studied in this work. The result showed that N-terminal cyclization occurred at the most of the glutamine(Q) or carbamoylmethyl-cysteine(CAM_C) residues and the reaction is often incomplete or partial, both types of peptides could often exist in its respective state at the same time, and the behavior of modified peptides in revered phase chromatography is also changed. The success rate of protein identification could be obviously improved if adding the N-terminal cyclization modification in the database searching. These results will be very helpful in the mass spectrometric data analysis of proteomic study.

Synthesis of new Imidazoline derives by the ethylene diamine cyclization from nitril compounds

The imidazol and imidazoline derives have many advantages in therapy. For the synthesis of new imidazoline derives, the authors implemented the ethylene diamene cyclization from nitril compounds differently substituted. Then, a dehydrogenation of these imidazolines leaded to the imidazol derives. The last reaction consisted of the condensation of these imidazolines and imidazoles. The authors subjected their synthesized products to an ophthalmic protective evaluation (anti-oedema, secondary anti-hyperemia) on the experimental ocular irritancy models

Synthesis of bicucullin by method of forming cycle with Bischler-Napieralski

Phthalideisoquinoline alcaloid norbicuculline have been synthesized by Bischler-Napieralski cyclization. The racemic norbicuculline were resolved into (+) and (-) forms. Methylation of (+ )norbicuculline provided (+)bicuculline. Researching bicucullin synthesis by the method of Bischler – napieralski shows that bicucullin is synthesized from Piperonal and Beta - (3,4 methylendioxy) – phenylethylamin by method of Bischler – napieralski. Isomers of ( ±) erythro and (±) threo norbicucullin are easily separated thank to nhờ sắc kí cột silicagel. Afterwards, optical isomers of (±) norbicucullin are also separated with high optical purity and good output. Methyl changed into (+) erythro norbicucullin is to gather (+) bicucullin gaining standards for biological experiments