Chemically Cross-Linked Hammerhead Ribozyme as an Efficient RNA Interference Tool.

RNA-cleaving ribozymes are promising candidates as general tools of RNA interference (RNAi) in gene manipulation. However, compared with other RNA systems, such as siRNA and CRISPR technologies, the ribozyme tools are still far from broad applications on RNAi due to their poor performance in the cellular context. In this work, we report an efficient RNAi tool based on chemically modified hammerhead ribozyme (HHR). By the introduction of an intramolecular linkage into the minimal HHR to reconstruct the distal interaction within the tertiary ribozyme structure, this cross-linked HHR exhibits efficient RNA substrate cleavage activities with almost no sequence constraint. Cellular experiments suggest that both exogenous and endogenous RNA expression can be dramatically knocked down by this HHR tool with levels comparable to those of siRNA. Unlike the widely applied protein-recruiting RNA systems (siRNA and CRISPR), this ribozyme tool functions solely on RNA itself with great simplicity, which may provide a new approach for gene manipulation in both fundamental and translational studies.

AgNPs-Modified Polylactic Acid Microneedles: Preparation and In Vivo/In Vitro Antimicrobial Studies.

OBJECTIVE: To prepare polylactic acid microneedles (PLAMNs) with sustained antibacterial effect to avoid skin infection caused by traditional MNs-based biosensors. METHODS: Silver nanoparticles (AgNPs) were synthesized using an in-situ reduction process with polydopamine (PDA). PLAMNs were fabricated using the hot-melt method. A series of pressure tests and puncture experiments were conducted to confirm the physicochemical properties of PLAMNs. Then AgNPs were modified on the surface of PLAMNs through in-situ reduction of PDA, resulting in the formation of PLAMNs@PDA-AgNPs. The in vitro antibacterial efficacy of PLAMNs@PDA-AgNPs was evaluated using agar diffusion assays and bacterial liquid co-culture approach. Wound healing and simulated long-term application were performed to assess the in vivo antibacterial effectiveness of PLAMNs@PDA-AgNPs. RESULTS: The MNs array comprised 169 tiny needle tips in pyramidal rows. Strength and puncture tests confirmed a 100% puncture success rate for PLAMNs on isolated rat skin and tin foil. SEM analysis revealed the integrity of PLAMNs@PDA-AgNPs with the formation of new surface substances. EDS analysis indicated the presence of silver elements on the surface of PLAMNs@PDA-AgNPs, with a content of 14.44%. Transepidermal water loss (TEWL) testing demonstrated the rapid healing of micro-pores created by PLAMNs@PDA-AgNPs, indicating their safety. Both in vitro and in vivo tests confirmed antibacterial efficacy of PLAMNs@PDA-AgNPs. CONCLUSIONS: In conclusion, the sustained antibacterial activity exhibited by PLAMNs@PDA-AgNPs offers a promising solution for addressing skin infections associated with MN applications, especially when compared to traditional MN-based biosensors. This advancement offers significant potential for the field of MN technology.

Harnessing Catalytic RNA Circuits for Construction of Artificial Signaling Pathways in Mammalian Cells.

Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR-Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA-based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis-related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression.

Integrating Ligands into Nucleic Acid Systems.

Signal transduction from non-nucleic acid ligands (small molecules and proteins) to structural changes of nucleic acids plays a crucial role in both biomedical analysis and cellular regulations. However, how to bridge between these two types of molecules without compromising the expandable complexity and programmability of the nucleic acid nanomachines is a critical challenge. Compared with the previously most widely applied transduction strategies, we review the latest advances of a kinetically controlled approach for ligand-oligonucleotide transduction in this Concept article. This new design works through an intrinsic conformational alteration of the nucleic acid aptamer upon the ligand binding as a governing factor for nucleic acid strand displacement reactions. The functionalities and applications of this transduction system as a ligand converter on biosensing and DNA computation are described and discussed. Furthermore, we propose some potential scenarios for utilization of this ligand transduction design to regulate gene expression through synthetic RNA switches in the cellular contexts. Finally, future perspectives regarding this ligand-oligonucleotide transduction platform are also discussed.

Programming Non-Nucleic Acid Molecules into Computational Nucleic Acid Systems.

Nucleic acid (NA) computation has been widely developed in the past years to solve kinds of logic and mathematic issues in both information technologies and biomedical analysis. However, the difficulty to integrate non-NA molecules limits its power as a universal platform for molecular computation. Here, we report a versatile prototype of hybridized computation integrated with both nucleic acids and non-NA molecules. Employing the conformationally controlled ligand converters, we demonstrate that non-NA molecules, including both small molecules and proteins, can be computed as nucleic acid strands to construct the circuitry with increased complexity and scalability, and can be even programmed to solve arithmetical calculations within the computational nucleic acid system. This study opens a new door for molecular computation in which all-NA circuits can be expanded with integration of various ligands, and meanwhile, ligands can be precisely programmed by the nuclei acid computation.

Non-classical cardenolides from Calotropis gigantea exhibit anticancer effect as HIF-1 inhibitors.

Six new non-classical cardenolides (1-6), and seventeen known ones (7-23) were isolated from Calotropis gigantea. All cardenolides showed inhibitory effect on hypoxia inducible factor-1 (HIF-1) transcriptional activity with IC50 of 8.85 nM-16.69 µM except 5 and 7. The novel 19-dihydrocalotoxin (1) exhibited a comparable HIF-1 inhibitory activity (IC50 of 139.57 nM) to digoxin (IC50 of 145.77 nM), a well-studied HIF-1 inhibitor, and 11, 12, 14, 16 and 19 presented 1.4-15.4 folds stronger HIF-1 inhibition than digoxin. 1 and 11 showed a dose-dependent inhibition on HIF-1α protein, which led to their HIF-1 suppressing effects. Compared with LO2 and H9c2 normal cell lines, both 1 and 11 showed selective cytotoxicity against various cancer cell lines including HCT116, HeLa, HepG2, A549, MCF-7, A2780 and MDA-MB-231. Moreover, a comprehensive structure-activity relationship was concluded for these non-classical cardenolides as HIF-1 inhibitors, which may shed some light on the rational design and development of cardenolide-based anticancer drugs.

Recent Methods for Purification and Structure Determination of Oligonucleotides.

Aptamers are single-stranded DNA or RNA oligonucleotides that can interact with target molecules through specific three-dimensional structures. The excellent features, such as high specificity and affinity for target proteins, small size, chemical stability, low immunogenicity, facile chemical synthesis, versatility in structural design and engineering, and accessible for site-specific modifications with functional moieties, make aptamers attractive molecules in the fields of clinical diagnostics and biopharmaceutical therapeutics. However, difficulties in purification and structural identification of aptamers remain a major impediment to their broad clinical application. In this mini-review, we present the recently attractive developments regarding the purification and identification of aptamers. We also discuss the advantages, limitations, and prospects for the major methods applied in purifying and identifying aptamers, which could facilitate the application of aptamers.

Application of chromatography technology in the separation of active alkaloids from Hypecoum leptocarpum and their inhibitory effect on fatty acid synthase.

A method that involved the combination of pH-zone-refining counter-current chromatography and semipreparative reversed-phase liquid chromatography has been established for the preparative separation of alkaloids from Hypecoum leptocarpum. From 1.2 g of crude sample, 31 mg N-feruloyltyramine, 27 mg oxohydrastinine, 47 mg hydroprotopine, 25 mg leptopidine, and 18 mg hypecocarpine have been obtained. The structure of the new compound, hypecocarpine, is confirmed based on the analysis of spectroscopic data, including NMR, UV, and IR spectroscopy and positive electrospray ionization mass spectrometry. The known chemical structures were characterized on the basis of (1) H and (13) C NMR spectroscopy. The purities of the five alkaloids are all over 92.7% as determined by high-performance liquid chromatography. The alkaloids' cytotoxicity in breast cancer cells is assessed by using a Cell Counting Kit assay and their inhibitory effect on fatty acid synthase expression is assessed by a Western blot assay. These results suggest that leptopidine could suppress growth and induce cytotoxicity in breast cancer cells and that the cytotoxicity of leptopidine may be related to its inhibitory effect on fatty acid synthase expression.

Performance of an immobilized microalgae-based process for wastewater treatment and biomass production: Nutrients removal, lipid induction, microalgae harvesting and dewatering.

Immobilized microalgae are good for wastewater treatment and biomass production. This study investigated treatment efficiency of a continuously operated system employing immobilized microalgae for secondary effluent of wastewater treatment plants, as well as the effectiveness on induction of valuable products, harvesting and dewatering of microalgae biomass. Under semi-continuous operation condition, microalgal dry weight increased 4.75 times within 2 d, associated with the highest removal rate of ammonia and phosphate at 28.95 mg/L·d and 4.83 mg/L·d, respectively. An immobilized microalgae membrane bioreactor (iMBR) was continuously operated for a month. The harvested immobilized microalgae beads were transferred to induction stage to obtain 4.5 times increase of lipid content per cell within 2 d. Immobilized microalgae performed 1.9 cm/s settling velocity and 97% water removal efficiency around 40 °C. A prospective integrated process on resource recovery and carbon neutrality was proposed for wastewater treatment, induction, harvesting and dewatering of immobilized microalgae cells.

Controllable DNA hybridization by host-guest complexation-mediated ligand invasion.

Dynamic regulation of nucleic acid hybridization is fundamental for switchable nanostructures and controllable functionalities of nucleic acids in both material developments and biological regulations. In this work, we report a ligand-invasion pathway to regulate DNA hybridization based on host-guest interactions. We propose a concept of recognition handle as the ligand binding site to disrupt Watson-Crick base pairs and induce the direct dissociation of DNA duplex structures. Taking cucurbit[7]uril as the invading ligand and its guest molecules that are integrated into the nucleobase as recognition handles, we successfully achieve orthogonal and reversible manipulation of DNA duplex dissociation and recovery. Moreover, we further apply this approach of ligand-controlled nucleic acid hybridization for functional regulations of both the RNA-cleaving DNAzyme in test tubes and the antisense oligonucleotide in living cells. This ligand-invasion strategy establishes a general pathway toward dynamic control of nucleic acid structures and functionalities by supramolecular interactions.

A kinetically controlled platform for ligand-oligonucleotide transduction.

Ligand-oligonucleotide transduction provides the critical pathway to integrate non-nucleic acid molecules into nucleic acid circuits and nanomachines for a variety of strand-displacement related applications. Herein, a general platform is constructed to convert the signals of ligands into desired oligonucleotides through a precise kinetic control. In this design, the ligand-aptamer binding sequence with an engineered duplex stem is introduced between the toehold and displacement domains of the invading strand to regulate the strand-displacement reaction. Employing this platform, we achieve efficient transduction of both small molecules and proteins orthogonally, and more importantly, establish logical and cascading operations between different ligands for versatile transduction. Besides, this platform is capable of being directly coupled with the signal amplification systems to further enhance the transduction performance. This kinetically controlled platform presents unique features with designing simplicity and flexibility, expandable complexity and system compatibility, which may pave a broad road towards nucleic acid-based developments of sophisticated transduction networks.

Controllable DNA strand displacement by independent metal-ligand complexation.

Introduction of artificial metal-ligand base pairs can enrich the structural diversity and functional controllability of nucleic acids. In this work, we revealed a novel approach by placing a ligand-type nucleoside as an independent toehold to control DNA strand-displacement reactions based on metal-ligand complexation. This metal-mediated artificial base pair could initiate strand invasion similar to the natural toehold DNA, but exhibited flexible controllability to manipulate the dynamics of strand displacement that was only governed by its intrinsic coordination properties. External factors that influence the intrinsic properties of metal-ligand complexation, including metal species, metal concentrations and pH conditions, could be utilized to regulate the strand dynamics. Reversible control of DNA strand-displacement reactions was also achieved through combination of the metal-mediated artificial base pair with the conventional toehold-mediated strand exchange by cyclical treatments of the metal ion and the chelating reagent. Unlike previous studies of embedded metal-mediated base pairs within natural base pairs, this metal-ligand complexation is not integrated into the nucleic acid structure, but functions as an independent toehold to regulate strand displacement, which would open a new door for the development of versatile dynamic DNA nanotechnologies.

A water-soluble nucleolin aptamer-paclitaxel conjugate for tumor-specific targeting in ovarian cancer.

Paclitaxel (PTX) is among the most commonly used first-line drugs for cancer chemotherapy. However, its poor water solubility and indiscriminate distribution in normal tissues remain clinical challenges. Here we design and synthesize a highly water-soluble nucleolin aptamer-paclitaxel conjugate (NucA-PTX) that selectively delivers PTX to the tumor site. By connecting a tumor-targeting nucleolin aptamer (NucA) to the active hydroxyl group at 2' position of PTX via a cathepsin B sensitive dipeptide bond, NucA-PTX remains stable and inactive in the circulation. NucA facilitates the uptake of the conjugated PTX specifically in tumor cells. Once inside cells, the dipeptide bond linker of NucA-PTX is cleaved by cathepsin B and then the conjugated PTX is released for action. The NucA modification assists the selective accumulation of the conjugated PTX in ovarian tumor tissue rather than normal tissues, and subsequently resulting in notably improved antitumor activity and reduced toxicity.

One Step to Separate Five Alkaloids from Hypecoum leptocarpum by High-Speed Counter-Current Chromatography.

High-speed counter-current chromatography (CCC) was firstly and successfully applied for the preparative separation and purification of alkaloids from crude extract of Hypecoum leptocarpum. After the measurement of partition coefficient of five target alkaloids in the two-phase solvent systems, the CCC was performed well with a two-phase solvent system composed of tetrachloromethane-chloroform-methanol-0.1 M HCl at a volume ratio of 1.5 : 2.5 : 3 : 2 (V/V/V/V). The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase. From 120 mg crude extract, 5 mg leptopidine, 32 mg oxohydrastinine, 27 mg (-)-N-methylanadine, 7 mg N-feruloyltyramine and 3 mg hypecoleptopine could be successfully separated. The amides alkaloid, N-feruloyltyramine, was firstly separated from H. leptocarpum. High-performance liquid chromatography analysis showed that the purity of each of the five target alkaloids was over 92%. Their chemical structures were confirmed by (1)H-NMR and (13)C-NMR data.

In vitro and in vivo biological activities of anthocyanins from Nitraria tangutorun Bobr. fruits.

Anthocyanins are the main compounds in Nitraria tangutorun Bobr. The enrichment and purification of anthocyanins on macroporous resins were investigated. Regarding anthocyanin purification, static adsorption and desorption were studied. The optimal experimental conditions were the following: resin type: X-5; static adsorption time: 6h; desorption solution: ethanol-water-HCl (80:19:1, V/V/V; pH 1); desorption time: 40min. Furthermore, the in vitro and in vivo biological activities of the anthocyanins were evaluated. The anthocyanins showed ideal scavenging effects on free radicals in vitro, especially on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl free radical (OH). In the animal experiment, blood lipid metabolism of hyperlipidemia rats was regulated by anthocyanin contents. The superoxide dismutase (SOD) activity and the total antioxidant capacity (TAC) of hyperlipidemia rats were also improved by anthocyanins. These results showed that anthocyanins from N. tangutorun Bobr. fruits had potential biological activities in vivo as well as in vitro.

Preparative Separation of N-Feruloyl Serotonin and N-(p-Coumaroyl) Serotonin from Safflower Seed Meal Using High-Speed Counter-Current Chromatography.

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of N-feruloyl serotonin (NF) and N-(p-coumaroyl) serotonin (NP) from safflower seed meal. After the measurement of partition coefficient of the two target compounds in the two-phase solvent systems, the HSCCC was performed well with a two-phase solvent system composed of CHCl3-methanol-0.1 M HCl at a volume ratio of 1 : 1 : 1, v/v. The upper phase was used as stationary phase and the lower phase was used as mobile phase. Under the optimized condition, 7.5 mg NF and 6.9 mg NP were separated from 40 mg crude sample with the purity of 98.8 and 97.3%, respectively. The structures of the isolated compounds were identified by (1)H NMR and (13)C NMR.

Preparative separation and purification of four cis-trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis-trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform-methanol-water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3mg of N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine (EEE), 4.4mg of N(1)(E)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (EZE), 7.2mg of N(1)(Z)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5mg of N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, (1)H NMR and (13)C NMR.