A Comparative Study on the DNA Interactions and Biological Activities of Benzophenanthridine and Protoberberine Alkaloids.

Numerous small molecules exert antitumor effects by interacting with DNA, thereby influencing processes, such as DNA replication, transcription, meiosis, and gene recombination. Benzophenanthridine and protoberberine alkaloids are known to bind DNA and exhibit many pharmacological activities. In this study, we conducted a comparative analysis of the interactions between these two classes of alkaloids with G-quadruplex (G4) DNA and double-stranded DNA (dsDNA). Protoberberine alkaloids showed a greater affinity for binding with G4s than with dsDNA, while benzophenanthridine alkaloids exhibited a significantly stronger binding capacity for dsDNA, especially in regions that are rich in AT base pairs. Benzophenanthridine alkaloids also exhibited much stronger toxicity to various cancer cells. Compared with protoberberine alkaloids, benzophenanthridine alkaloids displayed much stronger activity in inhibiting cellular DNA and RNA synthesis, arresting the cell cycle in the G2/M phase, inducing cell apoptosis, and leading to intracellular DNA damage. Given that dsDNA constitutes the predominant form of DNA within cells for the majority of the cell cycle, the significant antiproliferative activity of benzophenanthridine alkaloids could be attributed, in part, to their higher binding affinity for dsDNA, thereby exerting a more significant impact on cellular proliferation. These findings have valuable implications for understanding the biological activities of isoquinoline alkaloids and their antitumor applications.

A naphthalimide-based fluorescent platform for endoplasmic reticulum targeted imaging.

A series of naphthalimide dyes (TRNATR, MOTNAMOT, MPNAMP, TYNATY, PNAP and IZNAIZ) were designed and synthesized by altering the side chains of the naphthalimide. Without the need for ER-targeting groups, the first five dyes were found to specifically target the ER, likely due to their well-suited lipophilic properties. Furthermore, TRNATR and TYNATY were proven effective for studying ER stress, showing promise in tracking ER autophagy in living cells triggered by tunicamycin and nutritional starvation.

A Bispecific Chimeric Aptamer Design Platform Based on c-MET Aptamer with a Replaceable Redundant Region.

Molecular engineering enables the creation of aptamers with novel functions, but the prerequisite is a deep understanding of their structure and recognition mechanism. The cellular-mesenchymal epithelial transition factor (c-MET) is garnering significant attention due to the critical role of the c-MET/HGF signaling pathway in tumor development and invasion. This study reports a strategy for constructing novel chimeric aptamers that bind to both c-MET and other specific proteins. c-MET was identified to be the molecular target of a DNA aptamer, HF3-58, selected through cell-SELEX. The binding structure and mechanism of HF3-58 with c-MET were systematically studied, revealing the scaffold, recognition, and redundancy regions. Through molecular engineering design, the redundancy region was replaced with other aptamers possessing stem-loop structures, yielding novel chimeric aptamers with bispecificity for binding to c-MET and specific proteins. A chimeric bispecific aptamer HF-3b showed the ability to mediate the adhesion of T-cells to tumor cells, suggesting the prospective utility in tumor immunotherapy. These findings suggest that aptamer HF3-58 can serve as a molecular engineering platform for the development of diverse multifunctional ligands targeting c-MET. Moreover, comprehensive understanding of the binding mechanisms of aptamers will provide guidance for the design of functional aptamers, significantly expanding their potential applications.

Heptamethine Cyanine-Based Molecule Release Triggered by Mitochondrial ROS.

Conditionally activated molecule release in live cells would provide spatiotemporal control for the study and intervention of biological processes, e.g., bioactive molecule monitoring and controlled drug release. Mitochondria are the main sites of reactive oxygen species (ROS) production in cells. Here, we report an ROS-triggered molecule release strategy in mitochondria. A molecule IRTO with dual targeting groups was designed by covalently linking IR-780 (a mitochondrial targeted heptamethine cyanine) and 4-aminobutyl-thiazole orange (NH2-TO, a nuclear dye). IRTO diffused into live cells and first accumulated in mitochondria. As the cyanine moiety reacted with mitochondrial ROS directly or with the help of mitochondrial cytochromes, NH2-TO was released, escaped from mitochondria, and finally located in the nucleus. This process could be visualized by fluorescent imaging, i.e., red fluorescence (from the cyanine moiety of IRTO) first located in mitochondria, and green fluorescence (from NH2-TO) appeared and gradually enhanced in the nucleus with the increase of incubation time. The addition of H2O2 or lipopolysaccharide (LPS, an ROS accelerator) could accelerate the release of NH2-TO, whereas N-acetyl-l-cysteine (NAC, an ROS inhibitor) and mitoquinone mesylate (MitoQ, a mitochondrial ROS scavenger) could obviously decrease the release of NH2-TO. These results suggest that IRTO could serve as a fluorescent probe for monitoring ROS in mitochondria and that IR-780 might be a promising endogenous ROS-triggered molecule release platform.

DNA Aptamer Binding Octapeptide Repeat Region of Cellular Prion Protein.

Cellular prion protein (PrPC) is highly expressed in a variety of tumor cells and plays a crucial role in neurodegenerative diseases. Its N-terminal domain contains a conserved octapeptide (PHGGGWGQ) repeat sequence. The number of repeats has been correlated with the species as well as the development of associated diseases. Herein, PrPC was identified to be the molecular target of a high-affinity DNA aptamer HA5-68 obtained by cell-SELEX. Aptamer HA5-68 was further optimized to two short sequences (HA5-40-1 and HA5-40-2), and its binding site to PrPC was identified to be located in the loop-stem-loop region of the head of its secondary structure. HA5 series aptamers were demonstrated to bind the octapeptide repeat region of PrPC, as well as the synthesized peptides containing different numbers of octapeptide repeats. The PrPC expression on 42 cell lines was measured by using aptamer HA5-68 as a molecular probe. The clear understanding of the molecular structure and binding mechanism of this set of aptamers will provide information for the design of diagnostic methods and therapeutic drugs targeting PrPC.

Structural Optimization and Interaction Study of a DNA Aptamer to L1 Cell Adhesion Molecule.

The L1 cell adhesion molecule (L1CAM) plays important roles in the development and plasticity of the nervous system as well as in tumor formation, progression, and metastasis. New ligands are necessary tools for biomedical research and the detection of L1CAM. Here, DNA aptamer yly12 against L1CAM was optimized to have much stronger binding affinity (10-24 fold) at room temperature and 37 °C via sequence mutation and extension. This interaction study revealed that the optimized aptamers (yly20 and yly21) adopted a hairpin structure containing two loops and two stems. The key nucleotides for aptamer binding mainly located in loop I and its adjacent area. Stem I mainly played the role of stabilizing the binding structure. The yly-series aptamers were demonstrated to bind the Ig6 domain of L1CAM. This study reveals a detailed molecular mechanism for the interaction between yly-series aptamers and L1CAM and provides guidance for drug development and detection probe design against L1CAM.

A mitochondria-targeted fluorescent probe for imaging of endogenous carbon monoxide in living cells.

Carbon monoxide (CO), a vital gasotransmitter, plays critical functions in many physiological processes. Mitochondrial CO is closely related to mitochondrial respiration, thus the detection and imaging of mitochondrial CO in living cells is very important and has attracted much attention recently. In this paper, we developed a hemicyanine-based off-on fluorescent probe, CO-H1, which was used for monitoring endogenous mitochondrial CO levels in living cells. After reacted with CO in the presence of PdCl2, the fluorescence of CO-H1 was enhanced notably, accompanied by a significant red shift of absorption. CO-H1 exhibits low cytotoxicity, high sensitivity (detection limit of 0.048 µM), and good selectivity for CO. When incubated with living cells, probe CO-H1 mainly entered the mitochondria. CO-H1 was successfully applied to imaging the exogenous/endogenous mitochondrial CO in living cells, suggesting its potential application for further studying the biological functions of mitochondrial CO in living cells.

Generation of an Aptamer Targeting Receptor-Type Tyrosine-Protein Phosphatase F.

Cell-SELEX is a powerful tool to generate aptamers that specifically bind the native molecules on living cells. Here, we report an aptamer ZAJ4a generated by cell-SELEX. The molecular target of ZAJ4a was pulled down by the enriched cell-SELEX pool and identified to be the receptor-type tyrosine-protein phosphatase F (PTPRF) through a stable isotope labeling using amino acids in cell culture (SILAC)-based quantitative proteomic method. ZAJ4a showed high binding affinity with nanomolar range to cancer cells expressing PTPRF. Meanwhile, PTPRF was proven to highly express on several cancer cell lines using ZAJ4a as a molecular probe and to highly express in many kinds of cancer samples using gene expression profiling interactive analysis (GEPIA2) from the TCGA and GTEx databases. These results indicate that the aptamer generated by cell-SELEX showed good specificity at the molecular level. This cell-SELEX and target identification strategies show great potential for identifying biomarkers on the cell surface.

A photo-activated aptamer-drug conjugate for targeted drug delivery.

A photo-activated aptamer-drug conjugate, HG1-9-DNP, was developed based on an aptamer HG1-9 and a photolabile naphthalimide derivative DNP. HG1-9-DNP could be internalized into cells mediated by TfR, then photocleaved, and released a promising cytotoxic agent, DNNH, which arrested the cell cycle at the G2/M phase, resulting in high photo-induced cytotoxicity.

Potential G-quadruplexes within the Promoter Nuclease Hypersensitive Sites of the Heat-Responsive Genes in Rice.

G-quadruplexes (G4s) have been shown to be involved in the regulation of multiple cellular processes. Exploring putative G4-forming sequences (PQSs) in heat-responsive genes of rice and their folding structures under different conditions will help to understand the mechanism in response to heat stress. In this work, we discovered a prevalence of PQSs in nuclease hypersensitive sites within the promoters of heat-responsive genes. Moreover, 50 % of the searched G3 PQSs ((G3+ L1-7 )3+ G3+ ) locate in heat shock transcription factors. Circular dichroism spectroscopy, thermal difference spectroscopy, and UV melting analysis demonstrated the representative PQSs could adopt stable G4s at physiological temperature and potassium concentration. These PQSs were able to stall Klenow fragment (KF) DNA polymerase by the formation of G4s. However, the G4s with Tm values around 50-60 °C could be increasingly unwound by KF with the increase of temperatures from 25 to 50 °C, implying that these G4s could sense the changes in temperature by structural switch. This work offers fresh clues to understanding the potential of G4-involved functions of PQSs and the molecular events in plants in response to heat stress.

Characterization and Identification of Aptamers against CD49c for the Detection, Capture, and Release of Cancer Cells.

As a kind of recognition molecule, aptamers can be inserted into some regulatory sequences for the smart response of their targets. However, the molecular engineering might lead to the change of the binding affinity. Here, we present a stable aptamer ZAJ-2c and an environmentally sensitive aptamer ZAJ-2d optimized from an original cell-binding aptamer ZAJ-2, and the molecular target was further identified as CD49c on the cell membrane. ZAJ-2c was characterized with high binding ability independent of the presence of divalent cations at a temperature range from 4 to 37 °C, showing promise for measuring the expression of CD49c on cancer cells. Moreover, ZAJ-2d had a nanomolar binding affinity in the binding buffer at 4 °C, the same as ZAJ-2c, but lost the binding ability in a PBS buffer supplemented with 5 mM EDTA at 37 °C. This aptamer variant proved to selectively capture and release the CD49c positive cells by simply adjusting the temperatures and divalent cations. This set of aptamers might provide a toolbox for monitoring and operating of a wide range of cancer cells with CD49c expression on the surface, which will be helpful for the studying the heterogeneity of rare cells.

A mitochondria-targeted near-infrared fluorescent probe for detection and imaging of HSO3- in living cells.

Sulfur dioxide, an essential gas signaling molecule mainly produced in mitochondria, plays important roles in many physiological and pathological processes. Herein, a near-infrared fluorescent probe, A1, with good mitochondria targeting ability was developed for colorimetric and fluorescence detection of HSO3-. Probe A1 has a conjugated cyanine structure that can selectively react with HSO3- through the nucleophilic addition. The reaction with HSO3- destroys the conjugated structure of probe A1, resulting in fluorescence quenching, and accompaniedby color change of probe A1 solution from purple-red to colorless. Probe A1 showed high selectivity and good sensitivity to HSO3- in PBS. And the limit of detection was calculated to be 1.28 and 0.037 µM for colorimetry and fluorescence spectrophotometry respectively. In addition, probe A1 mainly entered the mitochondria in living cells, and was successfully used for imaging the exogenous/endogenous HSO3- in cells. These results suggest the potential applications of probe A1 in biological systems.

Aptamer-Based Cell Nucleus Imaging via Expansion Microscopy.

Expansion microscopy (ExM) is a newly developed technology in recent years that enables nanoscale imaging under conventional microscopes. Herein, we report an aptamer-based ExM imaging strategy. A nucleus-targeting aptamer Ch4-1 was chemically labeled with a dye and an acrydite at each end to perform the functions of molecular recognition, fluorescence reporting, and gel anchoring. After binding cell nucleus, the dual labeled aptamer Ac-Ch4-1-FAM directly participated in gelation and anchored in polyacrylamide gel. After expanding the gel, high-resolution imaging was achieved by confocal microscopy. Multicolor ExM imaging was also realized by combining Ac-Ch4-1-FAM, antibodies and fluorescent dyes. This aptamer-based ExM could clearly image the chromatin morphology at different mitotic stages. The expansion process is simple and the aptamer labeling is easy. The aptamer-based ExM holds great promise in super-resolution imaging of cells and tissues.

Transferrin receptor-mediated internalization and intracellular fate of conjugates of a DNA aptamer.

Aptamers have excellent specificity and affinity in targeting cell surface receptors, showing great potential in targeted delivery of drugs, siRNA, mRNA, and various nanomaterials with therapeutic function. A better insight of the receptor-mediated internalization process of aptameric conjugates could facilitate the design of new targeted drugs. In this paper, human transferrin receptor-targeted DNA aptamer (termed HG1-9)-fluorophore conjugates were synthesized to visualize the internalization, intracellular transport, and nano-environmental pH of aptameric conjugates. Unlike transferrin that showed high recycling rate and short duration time in cells, the synthetic aptameric conjugates continuously accumulated within cells at a relatively slower rate, besides recycling back to cell surface. After long incubation (≥2 h), only very small amounts of HG1-9 conjugates (approximately 5%) entered late endosomes or lysosomes, and more than 90% of internalized HG1-9 was retained in cellular vesicles (pH 6.0-6.8), escaping from degradation. And among the internalized HG1-9 conjugates, approximately 20% was dissociated from transferrin receptor. The lower recycling ratios of HG1-9 conjugates and their dissociation from receptors promote the accurate and efficient release of their loaded drugs. These results suggest that aptamer HG1-9 could be provided as a versatile tool for specific and effective delivery of diverse therapeutic payloads.

p-Aminostyryl thiazole orange derivatives for monitoring mitochondrial viscosity in live cells.

Viscosity of cell microenvironment plays a significant role in maintaining the normal life activities of cells. Particularly, the abnormal viscosity in mitochondria is closely associated with lots of diseases and cellular dysfunctions. Herein, we developed a group of p-aminostyryl thiazole orange derivatives with different amino side chains. These probes showed good fluorescence response to viscosity with twisted intramolecular charge transfer mechanism, among them, the probes with diethylamino (TOB), dibutylamino (TOC) and pyrrolidin (TOE) side chains showed better response to the viscosity with 78-fold, 55-fold, and 88-fold fluorescence enhancement in 95% glycerol solution respectively. TOB, TOC, and TOE could enter live cells and mainly located in mitochondria. Treatment HeLa cells with nystatin, lipopolysaccharide or oleic acid caused significant fluorescence enhancement of these probes, suggesting the good potential for monitoring the variation of mitochondrial viscosity, as well as for investigating the related physiological process of inflammation and lipid metabolism.

Cell-SELEX-based selection of ssDNA aptamers for specifically targeting BRAF V600E-mutated melanoma.

Malignant melanoma is regarded as the most aggressive form of skin cancer, and is responsible for most death caused by skin cancer. BRAF mutations occur in approximately 40-50% of melanomas, with V600E being the most common mutation. Testing for BRAF mutations and targeted therapy have markedly improved long-term survival for patients with BRAF-mutated melanoma. Here, we report two aptamers, CH1 and CH5 generated by Cell-SELEX, against BRAF V600E-mutated human melanoma cells A375. The two aptamers exhibited high affinity to target cells with low dissociation constants (Kd) in the nanomolar range. Furthermore, the binding of two aptamers to target cells was independent of incubation temperature, and their molecular targets were demonstrated to be membrane proteins on the cell surface. We also demonstrated that aptamer CH1 was able to bind to metastatic colorectal cancer cells harboring BRAF V600E mutation, indicating a relationship between aptamer CH1 and BRAF V600E-related metastatic cancer. Owing to the structure stability and high selectivity to BRAF V600E-mutated targeting cells, aptamer CH1 holds great potential as a molecular probe for the detection of BRAF V600E-mutated metastatic melanoma.

A DNA Aptameric Ligand of Human Transferrin Receptor Generated by Cell-SELEX.

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.

A 4-aminonaphthalimide-based fluorescent traceable prodrug with excellent photoinduced cytotoxicity.

A blue light activated anti-cancer prodrug, NST, was designed based on a photoactive 4-aminonaphthalimide derivative and an anticancer drug, 10-hydroxycamptothecin. NST was hard to be taken up by living cells and showed negligible dark cytotoxicity. The irradiation caused photocleavage of NST and resulted in high cytotoxicity.

Ratiometric detection and imaging of hydrogen sulfide in mitochondria based on a cyanine/naphthalimide hybrid fluorescent probe.

Hydrogen sulfide (H2S) in mitochondria plays important roles in many mitochondria-related physiological and pathological processes. Herein, a cyanine/naphthalimide hybrid fluorescent probe, L1, was designed for the ratiometric detection and imaging of mitochondrial H2S, in which cyanine and naphthalimide were used as the mitochondria-targeting group and H2S response group, respectively. Besides its good mitochondria-targeting ability, L1 also showed high sensitivity and good selectivity for H2S. Moreover, on the basis of the fluorescence ratio of naphthalimide to cyanine fluorophore, it was successfully applied to monitor the endogenous and exogenous mitochondrial H2S in live cells. Additionally, the endogenous mitochondrial H2S in different cell lines was measured by probe L1.

FnCas12a/crRNA assisted dumbbell-PCR detection of IsomiRs with terminal and inner sequence variants.

Herein, we report a FnCas12a/crRNA assisted Dumbbell-PCR method for the detection of isomiRs with double specificity and magnification. The single nucleotide variant of isomiRs in terminals and/or inner sequence could be discriminated by this strategy. Using this method, let-7a isomiRs in MCF-7 and MCF-7R cell lines were analyzed.