A Unique G-Quadruplex Aptamer: A Novel Approach for Cancer Cell Recognition, Cell Membrane Visualization, and RSV Infection Detection.

Surface staining has emerged as a rapid technique for applying external stains to trace cellular identities in diverse populations. In this study, we developed a distinctive aptamer with selective binding to cell surface nucleolin (NCL), bypassing cytoplasmic internalization. Conjugation of the aptamer with a FAM group facilitated NCL visualization on live cell surfaces with laser confocal microscopy. To validate the aptamer-NCL interaction, we employed various methods, including the surface plasmon resonance, IHC-based flow cytometry, and electrophoretic mobility shift assay. The G-quadruplex formations created by aptamers were confirmed with a nuclear magnetic resonance and an electrophoretic mobility shift assay utilizing BG4, a G-quadruplex-specific antibody. Furthermore, the aptamer exhibited discriminatory potential in distinguishing between cancerous and normal cells using flow cytometry. Notably, it functioned as a dynamic probe, allowing real-time monitoring of heightened NCL expression triggered by a respiratory syncytial virus (RSV) on normal cell surfaces. This effect was subsequently counteracted with dsRNA transfection and suppressed the NCL expression; thus, emphasizing the dynamic attributes of the probe. These collective findings highlight the robust versatility of our aptamer as a powerful tool for imaging cell surfaces, holding promising implications for cancer cell identification and the detection of RSV infections.

Unveiling the role of G-quadruplex structure in promoter region: Regulation of ABCA1 expression in macrophages possibly via NONO protein recruitment.

ABCA1 has been found to be critical for cholesterol efflux in macrophages. Understanding the mechanism regulating ABCA1 expression is important for the prevention and treatment of atherosclerosis. In the present study, a G-quadruplex (G4) structure was identified in the ABCA1 promoter region. This G4 was shown to be essential for ABCA1 transcription. Stabilizing the G4 by ligands surprisingly upregulated ABCA1 expression in macrophages. Knocking out the G4 remarkably reduced ABCA1 expression, and abolished the increase of ABCA1 expression induced by the G4 ligand. By pull-down assays, the protein NONO was identified as an ABCA1 G4 binder. Overexpression or repression of NONO significantly induced upregulation and downregulation of ABCA1 expression, respectively. ChIP and EMSA experiments showed that the G4 ligand promoted the binding between the ABCA1 G4 and NONO, which led to more recruitment of NONO to the promoter region and enhanced ABCA1 transcription. Finally, the G4 ligand was shown to significantly reduce the accumulation of cholesterol in macrophages. This study showed a new insight into the regulation of gene expression by G4, and provided a new molecular mechanism regulating ABCA1 expression in macrophages. Furthermore, the study showed a possible novel application of the G4 ligand: preventing and treating atherosclerosis.

Tetraphenylethene derivative that discriminates parallel G-quadruplexes.

G-Quadruplex (G4), as a non-canonical nucleic acid secondary structure, has been proved to be prevalent in genomes and plays important roles in many biological processes. Ligands targeting G4, especially small-molecular fluorescent light-up probes with selectivity for special conformations, are essential for studying the relationship between G4 folding and the cellular response. However, their development still remains challenging but is attracting massive attention. Here, we synthesized a new tetraphenylethene derivative, namely TPE-B, as a parallel G4 probe. Fluorescence experiments showed that TPE-B could give out a strong fluorescence response to the G4 structure. Moreover, it gave a much higher fluorescence intensity response to parallel G4s than anti-parallel ones, which indicated that TPE-B could serve as a special tool for probing parallel G4s. The circular dichroism (CD) spectra and melting curves showed that TPE-B could selectively bind and stabilize parallel G4s without changing their topology. ESI-MS studies showed that TPE-B could bind to parallel G4 with a 1 : 1 stoichiometry. The gel staining results showed that TPE-B was a good candidate for probing parallel G4s. Altogether, the TPE-B molecule may serve as a promising new probe that can discriminate parallel G4s.

A Small Ligand That Selectively Binds to the G-quadruplex at the Human Vascular Endothelial Growth Factor Internal Ribosomal Entry Site and Represses the Translation.

G-quadruplexes are believed to have important biological functions, so many small molecules have been screened or developed for targeting G-quadruplexes. However, it is still a major challenge to find molecules that recognize specific G-quadruplexes. Here, by using a combination of surface plasmon resonance, electrospray ionization mass spectrometry, circular dichroism, Western blot, luciferase assay, and reverse transcriptase stop assay, we observed a small molecule, namely, oxymatrine (OMT) that could selectively bind to the RNA G-quadruplex in 5'-untranslated regions (UTRs) of human vascular endothelial growth factor (hVEGF), but could not bind to other G-quadruplexes. OMT could selectively repress the translation of VEGF in cervical cancer cells. Furthermore, it could recognize VEGF RNA G-quadruplexes in special conformations. The results indicate that OMT may serve as a potentially special tool for studying the VEGF RNA G-quadruplex in cells and as a valuable scaffold for the design of ligands that recognize different G-quadruplexes.

Conformation of G-quadruplex Controlled by Click Reaction.

G-quadruplexes are non-canonical four stranded secondary structures possessing great biological importance. Controlling G-quadruplex conformation for further regulating biological processes is both exciting and challenging. In this study, we described a method for regulating G-quadruplex conformation by click chemistry for the first time. 8-ethynyl-2'-deoxyguanosine was synthesized and incorporated into a 12-nt telomere DNA sequence. Such a sequence, at first, formed mixed parallel/anti-parallel G-quadruplexes, while it changed to anti-parallel after reaction with azidobenzene. Meanwhile, the click reaction can give the sequence intense fluorescence.

2'-O-Methyl-8-methylguanosine as a Z-Form RNA Stabilizer for Structural and Functional Study of Z-RNA.

In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we developed a Z-form RNA stabilizer m8Gm, and demonstrated that incorporation of m8Gm into RNA can markedly stabilize the Z-RNA at low salt conditions. Using the m8Gm-contained Z-RNA, we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.

An intramolecular antiparallel G-quadruplex formed by human telomere RNA.

Until now, RNA G-quadruplexes were believed to only adopt a parallel G-quadruplex structure. In this study, we describe the first observation of an antiparallel RNA G-quadruplex formed by human telomere RNA. This newly described topology is of great interest as it shows that RNA G-quadruplexes can also be polymorphic and adopt structures that are different from the parallel configuration.

Antiparallel RNA G-quadruplex Formed by Human Telomere RNA Containing 8-Bromoguanosine.

In this study, by combining nuclear magnetic resonance (NMR), circular dichroism (CD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and gel electrophoresis, we report an unusual topological structure of the RNA G-quadruplex motif formed by human telomere RNA r(UAGGGU) containing 8-bromoguanosine. Results showed that the RNA sequence formed an antiparallel tetramolecular G-quadruplex, in which each pair of diagonal strands run in opposite directions. Furthermore, guanosines were observed both in syn- and anti-conformations. In addition, two of these G-quadruplex subunits were found to be stacking on top of each other, forming a dimeric RNA G-quadruplex. Our findings provide a new insight into the behavior of RNA G-quadruplex structures.

Unusual Topological RNA Architecture with an Eight-Stranded Helical Fragment Containing A-, G-, and U-Tetrads.

Human telomeric RNA performs various cellular functions such as telomere length regulation, heterochromatin formation, and chromosome end protection. Using a combination of nuclear magnetic resonance, circular dichroism, and gel electrophoresis, we observed an unusual topological structure formed by human telomere RNA r(GUUAGGGU). Our results showed that every set of four strands formed a parallel G-quadruplex as symmetry-related units containing four G-tetrads, two U-tetrads, and one A-tetrad. An eight-stranded helical fragment containing A-, G-, and U-tetrads provided a central intercalated scaffold that connected two G-quadruplex units in an alternating antiparallel arrangement, giving rise to a novel RNA architecture. This higher order RNA structure is so stable that it would be surprising if similar structures do not occur in nature. Our findings provide a new insight into the behavior of human telomeric RNA molecules.

Finding a human telomere DNA-RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: a protective structure for telomere end.

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA-RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA-RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA-RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA-RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA-RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.

Modified emulsion solvent evaporation method for fabricating core-shell microspheres.

Solvent evaporation/extraction method is widely used for core-shell microspheres fabrication. However, the solvent evaporation rate, as an essential factor for polymers phase separation, is difficult to control which results in failure of complete phase separation between polymers. At the present study, the selective dissolution technique was used to improve the phase separation, and successfully fabricate core-shell microspheres for controlled delivery of drug with reduced initial burst release. The core-shell microspheres were prepared with poly(l-lactic-co-glycolic acid) (PLGA) and poly(l-lactic acid) (PLLA), and aspirin was used as model drug. Ethyl acetate (EtAc) was applied to ameliorate the phase separation during the preparation process. The ratio of dichloromethane (DCM)/EtAc seriously affected the distribution of polymer molecules and the formation of the core-shell structure. The internal morphology of the microspheres varied depending on the amount of EtAc. Core-shell structure with a dense core of PLLA and a shell of PLGA was well formed when 2 ml EtAc was used. The differential scanning calorimeters (DSC) results showed two distinct melting points which confirmed a completely polymer phased separation occurred. These microspheres showed sustained release of aspirin for at least 456 h with a little burst release (3.49%).

[Preparation of sustained release microspheres containing oxymatrine and their release characteristics in vitro].

OBJECTIVE: To prepare poly(lactide-co-glycolide) (PLGA) microspheres containing Oxymatrine (OMT-PLGA-MS) and study their release characteristics in vitro. METHODS: OMT-PLGA-MS was prepared with PLGA as carriers using double emulsion solvent evaporation method (W/O/W). The preparation technology of microspheres was optimized by orthogonal design, and the microspheres were characterized in terms of morphology, the rate of drug loading, encapsulation efficiency, and in vitro drug release. RESULTS: The formed microspheres were spherical with smooth surfaces and the average size was (98.3 +/- 3.4) microm. The encapsulation efficiency and rate of drug loading were (70.14 +/- 3.47)% and (21.59 +/- 1.07)%, respectively. In vitro release study revealed that 84.63% of OMT was released from OMT-PLGA-MS in 240 hours, and the Higuchi model fitted OMT release pattern best. CONCLUSION: OMT-PLGA-MS is prepared successfully and shows good sustained release characteristics.