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1.
J Am Chem Soc ; 146(23): 15815-15824, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38832857

RESUMO

Ribonuclease targeting chimera (RIBOTAC) represents an emerging strategy for targeted therapy. However, RIBOTAC that is selectively activated by bio-orthogonal or cell-specific triggers has not been explored. We developed a strategy of inducible RIBOTAC (iRIBOTAC) that enables on-demand degradation of G-quadruplex (G4) RNAs for precision cancer therapy. iRIBOTAC is designed by coupling an RNA G4 binder with a caged ribonuclease recruiter, which can be decaged by a bio-orthogonal reaction, tumor-specific enzyme, or metabolite. A bivalent G4 binder is engineered by conjugating a near-infrared (NIR) fluorescence G4 ligand to a noncompetitive G4 ligand, conferring fluorescence activation on binding G4s with synergistically enhanced affinity. iRIBOTAC is demonstrated to greatly knockdown G4 RNAs upon activation under bio-orthogonal or cell-specific stimulus, with dysregulation of gene expressions involving cell killing, channel regulator activity, and metabolism as revealed by RNA sequencing. This strategy also shows a crucial effect on cell fate with remarkable biochemical hallmarks of apoptosis. Mice model studies demonstrate that iRIBOTAC allows selective imaging and growth suppression of tumors with bio-orthogonal and tumor-specific controls, highlighting G4 RNA targeting and inducible silencing as a valuable RIBOTAC paradigm for cancer therapy.


Assuntos
Quadruplex G , RNA Mensageiro , Ribonucleases , Humanos , Animais , Camundongos , Ribonucleases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Inativação Gênica , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Neoplasias/genética
2.
Anal Chim Acta ; 1312: 342764, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38834269

RESUMO

BACKGROUND: Osteopontin (OPN) is closely associated with tumorigenesis, growth, invasion, and immune escape and it serves as a plasma biomarker for hepatocellular carcinoma (HCC). Nevertheless, the accurate and rapid detection of low-abundance OPN still poses significant challenges. Currently, the majority of protein detection methods rely heavily on large precision instruments or involve complex procedures. Therefore, developing a simple, enzyme-free, rapid colorimetric analysis method with high sensitivity is imperative. RESULTS: In this study, we have developed a portable colorimetric biosensor by integrating the triple-helix aptamer probe (THAP) and catalytic hairpin assembly (CHA) strategy, named as T-CHA. After binding to the OPN, the trigger probe can be released from THAP, then initiates the CHA reaction and outputs the signal through the formation of a G-quadruplex/Hemin DNAzyme with horseradish peroxidase-like activity. Consequently, this colorimetric sensor achieves visual free-labeled detection without additional fluorophore modification and allows for accurate quantification by measuring the optical density of the solution at 650 nm. Under optimal conditions, the logarithmic values of various OPN concentrations exhibit satisfactory linearity in the range of 5 pg mL-1 to 5 ng mL-1, with a detection limit of 2.04 pg mL-1. Compared with the widely used ELISA strategy, the proposed T-CHA strategy is rapid (∼105 min), highly sensitive, and cost-effective. SIGNIFICANCE: The T-CHA strategy, leveraging the low background leakage of THAP and the high catalytic efficiency of CHA, has been successfully applied to the detection of OPN in plasma, demonstrating significant promise for the early diagnosis of HCC in point-of-care testing. Given the programmability of DNA and the universality of T-CHA, it can be readily modified for analyzing other useful tumor biomarkers.


Assuntos
Aptâmeros de Nucleotídeos , Colorimetria , Osteopontina , Colorimetria/métodos , Aptâmeros de Nucleotídeos/química , Humanos , Osteopontina/sangue , Osteopontina/química , Osteopontina/análise , Técnicas Biossensoriais/métodos , DNA Catalítico/química , DNA Catalítico/metabolismo , Limite de Detecção , Quadruplex G
3.
Comput Biol Med ; 177: 108683, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38838555

RESUMO

G-Quadruplex DNA (GQ-DNA) is one of the most important non-canonical nucleic acid structures. GQ-DNA forming sequences are present in different crucial genomic regions and are abundant in promoter regions of several oncogenes. Therefore, GQ-DNA is an important target for anticancer drugs and hence binding interactions between GQ-DNA and small molecule ligands are of great importance. Since GQ-DNA is a highly polymorphic structure, it is important to identify ligand molecules which preferentially target a particular quadruplex sequence. In this present study, we have used a FDA approved drug called imatinib mesylate (ligand) which is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia, gastrointestinal stromal tumours. Different spectroscopic techniques as well as molecular docking investigations and molecular simulations have been used to explore the interaction between imatinib mesylate with VEGF GQ DNA structures along with duplex DNA, C-Myc, H-Telo GQ DNA. We found that imatinib mesylate shows preferential interaction towards VEGF GQ DNA compared to C-Myc, H-Telo GQ and duplex DNA. Imatinib mesylate seems to be an efficient ligand for VEGF GQ DNA, suggesting that it might be used to regulate the expression of genes in cancerous cells.


Assuntos
Antineoplásicos , Quadruplex G , Mesilato de Imatinib , Simulação de Acoplamento Molecular , Fator A de Crescimento do Endotélio Vascular , Mesilato de Imatinib/uso terapêutico , Mesilato de Imatinib/química , Mesilato de Imatinib/farmacologia , Quadruplex G/efeitos dos fármacos , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , DNA/química , DNA/metabolismo
4.
Anal Chem ; 96(22): 9097-9103, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38768044

RESUMO

Herein, a fluorescence light-up 3D DNA walker (FLDW) was powered and accelerated by endogenous adenosine-5'-triphosphate (ATP) molecules to construct a biosensor for sensitive and rapid label-free detection and imaging of microRNA-221 (miRNA-221) in malignant tumor cells. Impressively, ATP as the driving force and accelerator for FLDW could significantly accelerate the operation rate of FLDW, reduce the likelihood of errors in signaling, and improve the sensitivity of detection and imaging. When FLDW was initiated by output DNA H1-op transformed by target miRNA-221, G-rich sequences in the S strand, anchored to AuNP, were exposed to form G-quadruplexes (G4s), and thioflavin T (ThT) embedded in the G4s emitted intense fluorescence to realize sensitive and rapid detection of target miRNA-221. Meanwhile, the specific binding of ThT to G4 with a weak background fluorescence response was utilized to enhance the signal-to-noise ratio of the label-free assay straightforwardly and cost-effectively. The proposed FLDW system could realize sensitive detection of the target miRNA-221 in the range of 1 pM to 10 nM with a detection limit of 0.19 pM by employing catalytic hairpin assembly (CHA) to improve the conversion of the target. Furthermore, by harnessing the abundant ATP present in the tumor microenvironment, FLDW achieved rapid and accurate imaging of miRNA-221 in cancer cells. This strategy provides an innovative and high-speed label-free approach for the detection and imaging of biomarkers in cancer cells and is expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.


Assuntos
Trifosfato de Adenosina , Técnicas Biossensoriais , DNA , MicroRNAs , MicroRNAs/análise , MicroRNAs/metabolismo , Humanos , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , DNA/química , Técnicas Biossensoriais/métodos , Imagem Óptica , Quadruplex G , Fluorescência , Corantes Fluorescentes/química , Limite de Detecção , Ouro/química
5.
Anal Chem ; 96(22): 9244-9253, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38773697

RESUMO

Sensitive identification and effective inactivation of the virus are paramount for the early diagnosis and treatment of viral infections to prevent the risk of secondary transmission of viruses in the environment. Herein, we developed a novel two-step fluorescence immunoassay using antibody/streptavidin dual-labeled polystyrene nanobeads and biotin-labeled G-quadruplex/hemin DNAzymes with peroxidase-mimicking activity for sensitive quantitation and efficient inactivation of living Zika virus (ZIKV). The dual-labeled nanobeads can specifically bind ZIKV through E protein targeting and simultaneously accumulate DNAzymes, leading to the catalytic oxidation of Amplex Red indicators and generation of intensified aggregation-induced emission fluorescence signals, with a detection limit down to 66.3 PFU/mL and 100% accuracy. Furthermore, robust reactive oxygen species generated in situ by oxidized Amplex Red upon irradiation can completely kill the virus. This sensitive and efficient detection-inactivation integrated system will expand the viral diagnostic tools and reduce the risk of virus transmission in the environment.


Assuntos
DNA Catalítico , Zika virus , DNA Catalítico/química , DNA Catalítico/metabolismo , Imunoensaio/métodos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Limite de Detecção , Quadruplex G , Inativação de Vírus/efeitos da radiação , Humanos
6.
Adv Immunol ; 161: 109-126, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38763699

RESUMO

Besides the canonical B-form, DNA also adopts alternative non-B form conformations which are highly conserved in all domains of life. While extensive research over decades has centered on the genomic functions of B-form DNA, understanding how non-B-form conformations influence functional genomic states remains a fundamental and open question. Recent studies have ascribed alternative DNA conformations such as G-quadruplexes and R-loops as important functional features in eukaryotic genomes. This review delves into the biological importance of alternative DNA structures, with a specific focus on hematopoiesis and adaptive immunity. We discuss the emerging roles of G-quadruplex and R-loop structures, the two most well-studied alternative DNA conformations, in the hematopoietic compartment and present evidence for their functional roles in normal cellular physiology and associated pathologies.


Assuntos
Imunidade Adaptativa , Quadruplex G , Hematopoese , Humanos , Hematopoese/genética , Animais , DNA/imunologia , Conformação de Ácido Nucleico
7.
Anal Chim Acta ; 1308: 342649, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38740457

RESUMO

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-powered biosensor with a G-quadruplex (G4) reporter offer the benefits of simplicity and sensitivity, making them extensively utilized in detection applications. However, these biosensors used for monitoring pollutants in environmental water samples may face the problem of high background signal and easy interference due to the "signal-off" output. It is obvious that a biosensor based on the CRISPR/Cas12a system and G4 with a "signal on" output mode needs to be designed for detecting environmental pollutants. RESULTS: By using phosphorothioate-modified G4 as a reporter and catalytic hairpin assembly (CHA) integrated with Cas12a as an amplification strategy, a "signal-on" colorimetric/photothermal biosensor (psG4-CHA/Cas) for portable detection of environmental pollutants was developed. With the help of functional nucleotides, the target pollutant (kanamycin or Pb2+) triggers a CHA reaction to produce numerous double-strand DNA, which can activate Cas12a's trans-cleavage activity. The active Cas12a cleaves locked DNA to release caged psG-rich sequences. Upon binding hemin, the psG-rich sequence forms a psG4/hemin complex, facilitating the oxidation of the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into the blue photothermal agent (oxTMB). The smartphone was employed for portable colorimetric detection of kanamycin and Pb2+. The detection limits were found to be 100 pM for kanamycin and 50 pM for Pb2+. Detection of kanamycin and Pb2+ was also carried out using a portable thermometer with a detection limit of 10 pM for kanamycin and 8 pM for Pb2+. SIGNIFICANCE: Sensitive, selective, simple and robust detection of kanamycin and Pb2+ in environmental water samples is achieved with the psG4-CHA/Cas system. This system not only provides a new perspective on the development of efficient CRISPR/Cas12a-based "signal-on" designs, but also has a promising application for safeguarding human health and environmental monitoring.


Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Quadruplex G , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , Colorimetria , Chumbo/análise , Poluentes Ambientais/análise , Limite de Detecção , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Poluentes Químicos da Água/análise , Proteínas de Bactérias , Endodesoxirribonucleases
8.
Anal Methods ; 16(20): 3220-3230, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38717230

RESUMO

Tuberculosis caused by Mycobacterium bovis poses a global infectious threat to humans and animals. Therefore, there is an urgent need to develop a sensitive, precise, and easy-to-readout strategy. Here, a novel tandem combination of a CRISPR/Cas12a system with dual HCR (denoted as CRISPR/Cas12a-D-HCR) was constructed for detecting Mycobacterium bovis. Based on the efficient trans-cleavage activity of the active CRISPR/Cas12a system, tandem-dsDNA with PAM sites was established using two flexible hairpins, providing multiple binding sites with CRISPR/Cas12a for further amplification. Furthermore, the activation of Cas12a initiated the second hybridization chain reaction (HCR), which integrated complete G-quadruplex sequences to assemble the hemin/G-quadruplex DNAzyme. With the addition of H2O2 and ABTS, a colorimetric signal readout strategy was achieved. Consequently, CRISPR/Cas12a-D-HCR achieved a satisfactory detection linear range from 20 aM to 50 fM, and the limit of detection was as low as 2.75 aM with single mismatched recognition capability, demonstrating good discrimination of different bacterial species. Notably, the practical application performance was verified via the standard addition method, with the recovery ranging from 96.0% to 105.2% and the relative standard deviations (RSD) ranging from 0.95% to 6.45%. The proposed CRISPR/Cas12a-D-HCR sensing system served as a promising application for accurate detection in food safety and agricultural fields.


Assuntos
Sistemas CRISPR-Cas , Colorimetria , Quadruplex G , Mycobacterium bovis , Mycobacterium bovis/genética , Sistemas CRISPR-Cas/genética , Colorimetria/métodos , Hibridização de Ácido Nucleico/métodos , Limite de Detecção , Animais , DNA Catalítico/química , Técnicas Biossensoriais/métodos , Proteínas Associadas a CRISPR/genética , DNA Bacteriano/genética
9.
Bioorg Chem ; 148: 107475, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38772293

RESUMO

The applications of antisense oligonucleotides (ASOs) in rare or common diseases treatment have garnered great attention in recent years. Nevertheless, challenges associated with stability and bioavailability still persist, hampering the efficiency of ASOs. This work presents an ASO prodrug with parallel G-quadruplex assembly and lysosome escape capabilities for oncotherapy. Our findings revealed that the end-assembled quadruplex structure effectively shielded the ASO from enzymatic degradation. Meanwhile, the conjugation of maleimide within the quadruplex enhanced cellular uptake, potentially offering an alternative cell entry mechanism that circumvents lysosome involvement. Notably, an optimized molecule, Mal2-G4-ASO, exhibited remarkable therapeutic effects both in vitro and in vivo. This work presents a promising avenue for enhancing the activity of nucleic acid drugs in oncotherapy and potentially other disease contexts.


Assuntos
Quadruplex G , Lisossomos , Oligonucleotídeos Antissenso , Pró-Fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/síntese química , Quadruplex G/efeitos dos fármacos , Humanos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/síntese química , Lisossomos/metabolismo , Animais , Estrutura Molecular , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Camundongos , Ensaios de Seleção de Medicamentos Antitumorais , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Camundongos Nus , Camundongos Endogâmicos BALB C
10.
Anal Chem ; 96(23): 9636-9642, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38808501

RESUMO

Organophosphate pesticides (OPs) are widely utilized in agricultural production, and the residues threaten public health and environmental safety due to their toxicity. Herein, a novel and simple DNA aptamer-based sensor has been fabricated for the rapid, visual, and quantitative detection of profenofos and isocarbophos. The proposed DNA aptamers with a G-quadruplex spatial structure could be recognized by SYBR Green I (SG-I), resulting in strong green fluorescence emitted by SG-I. The DNA aptamers exhibit a higher specific binding ability to target OP molecules through aromatic ring stacking, disrupting the interaction between SG-I and DNA aptamers to induce green fluorescence quenching. Meanwhile, the fluorescence wavelength of G-quadruplex fluorescence emission peaks changes, accompanied by an obvious fluorescence variation from green to blue. SG-I-modified aptasensor without any additive reference fluorescence units for use in multicolor fluorescence assay for selective monitoring of OPs was first developed. The developed aptasensor provides a favorable linear range from 0 to 200 nM, with a low detection limit of 2.48 and 3.01 nM for profenofos and isocarbophos, respectively. Moreover, it offers high selectivity and stability in real sample detection with high recoveries. Then, a self-designed portable smartphone sensing platform was successfully used for quantitative result outputs, demonstrating experience in designing a neotype sensing strategy for point-of-care pesticide monitoring.


Assuntos
Aptâmeros de Nucleotídeos , Benzotiazóis , Diaminas , Corantes Fluorescentes , Compostos Orgânicos , Praguicidas , Quinolinas , Espectrometria de Fluorescência , Aptâmeros de Nucleotídeos/química , Quinolinas/química , Praguicidas/análise , Diaminas/química , Corantes Fluorescentes/química , Benzotiazóis/química , Compostos Orgânicos/química , Técnicas Biossensoriais/métodos , Limite de Detecção , Quadruplex G , Malation/análogos & derivados
11.
Int J Biol Macromol ; 270(Pt 1): 132244, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38729459

RESUMO

To combat cancer, a comprehensive understanding of the molecular mechanisms and behaviors involved in carcinogenesis is crucial, as tumorigenesis is a complex process influenced by various genetic events and disease hallmarks. The B-MYB gene encodes a transcription factor involved in cell cycle regulation, survival, and differentiation in normal cells. B-MYB can be transformed into an oncogene through mutations, and abnormal expression of B-MYB has been identified in various cancers, including lung cancer, and is associated with poor prognosis. Targeting this oncogene is a promising approach for anti-cancer drug design. B-MYB has been deemed undruggable in previous reports, necessitating the search for novel therapeutic options. In this study, we found that the B-MYB gene promoter contains several G/C rich motifs compatible with G-quadruplex (G4) formation. We investigated and validated the existence of G4 structures in the promoter region of B-MYB, first in vitro using a combination of bioinformatics, biophysical, and biochemical methods, then in cell with the recently developed G4access method.


Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Regiões Promotoras Genéticas/genética , Humanos , Transativadores/genética , Transativadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Motivos de Nucleotídeos/genética
12.
Dalton Trans ; 53(23): 9700-9714, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38775704

RESUMO

Silver compounds are mainly studied as antimicrobial agents, but they also have anticancer properties, with the latter, in some cases, being better than their gold counterparts. Herein, we analyse the first example of a new Ag(I)-biscarbene that can bind non-canonical structures of DNA, more precisely G-quadruplexes (G4), with different binding signatures depending on the type of G4. Moreover, we show that this Ag-based carbene binds the i-motif DNA structure. Alternatively, its Au(I) counterpart, which was investigated for comparison, stabilises mitochondrial G4. Theoretical in silico studies elucidated the details of different binding modes depending on the geometry of G4. The two complexes showed increased cytotoxic activity compared to cisplatin, overcoming its resistance in ovarian cancer. The binding of these new drug candidates with other relevant biosubstrates was studied to afford a more complete picture of their possible targets. In particular, the Ag(I) complex preferentially binds DNA structures over RNA structures, with higher binding constants for the non-canonical nucleic acids with respect to natural calf thymus DNA. Regarding possible protein targets, its interaction with the albumin model protein BSA was also tested.


Assuntos
DNA , Quadruplex G , Prata , DNA/química , Prata/química , Humanos , Metano/química , Metano/análogos & derivados , Antineoplásicos/química , Antineoplásicos/farmacologia , Bovinos , Animais , Soroalbumina Bovina/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Corantes Fluorescentes/química
13.
Spectrochim Acta A Mol Biomol Spectrosc ; 318: 124489, 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-38788507

RESUMO

G-quadruplexs (G4s), four-stranded nucleic acid secondary structures, which formed by guanine-rich sequences play a vital role in human biological systems. Studies have shown that the formation of G4s is closely related to tumor development and apoptosis, which is considered as a new target for the development of anti-tumor drugs. Therefore, it is important to develop novel probes for G4s imaging. In this article, we engineered a near-infrared fluorescent probe (TOH) which can be activated by DNA G4s in living cells and tumor. TOH exhibits high selectivity to the structure of DNA G4s with the limit of detection for DNA G4s (Mito-0.5-2) is calculated to be 0.43 nM. Imaging studies of different cell lines revealed that the brighter fluorescence in cancer cell lines than in normal, indicating that DNA G4s maybe highly express in tumor cell lines. Simultaneously, TOH is also introduced into live tumor tissue imaging and found that the fluorescence intensity of tumor is the brightest relative to normal tissue, further validating the high expression of DNA G4s structures in tumor tissue. These features demonstrate TOH not only have the ability to image DNA G4 structures in real time, but also may have tumor diagnostic capabilities.


Assuntos
DNA , Corantes Fluorescentes , Quadruplex G , Humanos , Corantes Fluorescentes/química , DNA/química , Linhagem Celular Tumoral , Animais , Espectrometria de Fluorescência , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos
14.
Chem Biol Interact ; 395: 111031, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38703805

RESUMO

Alternative DNA structures play critical roles in fundamental biological processes linked to human diseases. Thus, targeting and stabilizing these structures by specific ligands could affect the progression of cancer and other diseases. Here, we describe, using methods of molecular biophysics, the interactions of two oxidatively locked [Co2L3]6+ cylinders, rac-2 and meso-1, with diverse alternative DNA structures, such as junctions, G quadruplexes, and bulges. This study was motivated by earlier results demonstrating that both Co(III) cylinders exhibit potent and selective activity against cancer cells, accumulate in the nucleus of cancer cells, and prove to be efficient DNA binders. The results show that the bigger cylinder rac-2 stabilizes all DNA structures, while the smaller cylinder meso-1 stabilizes just the Y-shaped three-way junctions. Collectively, the results of this study suggest that the stabilization of alternative DNA structures by Co(III) cylinders investigated in this work might contribute to the mechanism of their biological activity.


Assuntos
Cobalto , DNA , DNA/química , DNA/metabolismo , Cobalto/química , Humanos , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Conformação de Ácido Nucleico , Quadruplex G
15.
Nat Commun ; 15(1): 3963, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38729943

RESUMO

Translation initiation in bacteria is frequently regulated by various structures in the 5' untranslated region (5'UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aim to explore how G4 formation in mRNA (RG4) at 5'UTR impacts translation using a T7-based in vitro translation system and in E. coli. We show that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We find that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We propose a physical barrier model in which bulky structures in 5'UTR biases ribosome movement toward the downstream start codon, thereby increasing the translation output. This study provides biophysical insights into the regulatory role of 5'UTR structures in in vitro and bacterial translation, highlighting their potential applications in tuning gene expression.


Assuntos
Regiões 5' não Traduzidas , Escherichia coli , Quadruplex G , Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Regiões 5' não Traduzidas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Conformação de Ácido Nucleico , Estabilidade de RNA , Sítios de Ligação
16.
J Am Chem Soc ; 146(20): 13709-13713, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38738955

RESUMO

G-Quadruplex (G4) structures formed by guanine-rich DNA and RNA sequences are implicated in various biological processes. Understanding the mechanisms by which proteins recognize G4 structures is crucial for elucidating their functional roles. Here we present the X-ray crystal structure of an ankyrin protein bound to a parallel G4 structure. Our findings reveal a new specific recognition mode in which a bundle of α-helices and loops of the ankyrin form a flat surface to stack on the G-tetrad core. The protein employs a combination of hydrogen bonds and hydrophobic contacts to interact with the G4, and electrostatic interaction is used to enhance the binding affinity. This binding mechanism provides valuable insights into understanding G4 recognition by proteins.


Assuntos
Anquirinas , Quadruplex G , Modelos Moleculares , Anquirinas/química , Cristalografia por Raios X , Humanos , Ligação Proteica , Ligação de Hidrogênio
17.
Eur J Med Chem ; 274: 116536, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38805936

RESUMO

G-quadruplexes (G4s) are commonly formed in the G-rich strand of telomeric DNA. Ligands targeting telomeric G4 induce DNA damage and telomere dysfunction, which makes them potential antitumor drugs. New telomeric G4 ligands with drug-likeness are still needed to be exploited, especially with their antitumor mechanisms thoroughly discussed. In this study, a novel series of quinoxaline analogs were rationally designed and synthesized. Among them, R1 was the most promising ligand for its cytotoxic effects on tumor cells and stabilizing ability with telomeric G4. Cellular assays illustrated that R1 stabilized G4 and induced R-loop accumulation in the telomeric regions, subsequently triggering DNA damage responses, cell cycle arrest in G2/M phase, apoptosis and antiproliferation. Moreover, R1 evoked immunogenic cell death (ICD) in tumor cells, which promoted the maturation of bone marrow derived dendritic cells (BMDCs). In breast cancer mouse model, R1 exhibited a significant decrease in tumor burden through the immunomodulatory effects, including the increase of CD4+ and CD8+ T cells in tumors and cytokine levels in sera. Our research provides a new idea that targeting telomeric G4 induces DNA damage responses, causing antitumor effects both in vitro and in vivo, partially due to the enhancement of immunomodulation.


Assuntos
Antineoplásicos , Proliferação de Células , Quadruplex G , Quinoxalinas , Telômero , Quadruplex G/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Quinoxalinas/química , Quinoxalinas/farmacologia , Quinoxalinas/síntese química , Animais , Humanos , Telômero/efeitos dos fármacos , Ligantes , Camundongos , Proliferação de Células/efeitos dos fármacos , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Relação Estrutura-Atividade , Relação Dose-Resposta a Droga , Feminino , Imunomodulação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Dano ao DNA/efeitos dos fármacos
18.
J Biotechnol ; 390: 39-49, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38740306

RESUMO

The TFE3 fusion gene, byproduct of Xp11.2 translocation, is the diagnostic marker for translocation renal cell carcinoma (tRCC). Absence of any clinically recognized therapy for tRCC, pressing a need to create novel and efficient therapeutic approaches. Previous studies shown that stabilization of the G-quadruplex structure in oncogenes suppresses their expression machinery. To combat the oncogenesis caused by fusion genes, our objective is to locate and stabilize the G-quadruplex structure within the PRCC-TFE3 fusion gene. Using the Quadruplex-forming G Rich Sequences (QGRS) mapper and the Non-B DNA motif search tool (nBMST) online server, we found putative G-quadruplex forming sequences (PQS) in the PRCC-TFE3 fusion gene. Circular dichroism demonstrating a parallel G-quadruplex in the targeted sequence. Fluorescence and UV-vis spectroscopy results suggest that pyridostatin binds to this newly discovered G-quadruplex. The PCR stop assay, as well as transcriptional or translational inhibition using real time PCR and Dual luciferase assay, revealed that stable G-quadruplex formation affects biological processes. Confocal microscopy of HEK293T cells transfected with the fusion transcript confirmed G-quadruplexes formation in cell. This investigation may shed light on G-quadruplex's functions in fusion genes and may help in the development of therapies specifically targeted against fusion oncogenes, which would enhance the capability of current tRCC therapy approach.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Carcinoma de Células Renais , Quadruplex G , Neoplasias Renais , Proteínas de Fusão Oncogênica , Translocação Genética , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/tratamento farmacológico , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Neoplasias Renais/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteínas de Fusão Oncogênica/genética , Células HEK293 , Dicroísmo Circular , Aminoquinolinas , Proteínas de Neoplasias , Ácidos Picolínicos , Proteínas de Ciclo Celular
19.
Biosens Bioelectron ; 260: 116435, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-38820724

RESUMO

Electrochemical detection of miRNA biomarkers in complex physiological samples holds great promise for accurate evaluation of tumor burden in the perioperative period, yet limited by reproducibility and bias issues. Here, nanosensors installed with hybrid probes that responsively release catalytic DNAzymes (G-quadruplexes/hemin) were developed to solve the fidelity challenge in an immobilization-free detection. miRNA targets triggered toehold-mediated strand displacement reactions on the sensor surface and resulted in amplified shedding of DNAzymes. Subsequently, the interference background was removed by Fe3O4 core-facilitated magnetic separation. Binding aptamers of the electrochemical reporter (dopamine) were tethered closely to the catalytic units for boosting H2O2-mediated oxidation through proximity catalysis. The one-to-many conversion by dual amplification from biological-chemical catalysis facilitated sufficient homogeneous sensing signals on electrodes. Thereby, the nanosensor exhibited a low detection limit (2.08 fM), and high reproducibility (relative standard deviation of 1.99%). Most importantly, smaller variations (RSD of 0.51-1.04%) of quantified miRNAs were observed for detection from cell lysates, multiplexed detection from unprocessed serum, and successful discrimination of small upregulations in lysates of tumor tissue samples. The nanosensor showed superior diagnostic performance with an area under curve (AUC) of 0.97 and 94% accuracy in classifying breast cancer patients and healthy donors. These findings demonstrated the synergy of signal amplification and interference removal in achieving high-fidelity miRNA detection for practical clinical applications.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , Técnicas Eletroquímicas , Limite de Detecção , MicroRNAs , Humanos , MicroRNAs/isolamento & purificação , Técnicas Eletroquímicas/métodos , DNA Catalítico/química , Catálise , Quadruplex G , Neoplasias da Mama , Peróxido de Hidrogênio/química , Aptâmeros de Nucleotídeos/química , Feminino , Hemina/química , Reprodutibilidade dos Testes , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética
20.
Talanta ; 276: 126260, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38759364

RESUMO

Lead ion pollution has become a serious public health concern worldwide. Therefore, sensitive detection of Pb2+ is critical to control lead pollution, assess risks, and safeguard the health of vulnerable populations. This study reports a highly sensitive labelling-free electrochemical aptasensor for Pb2+ detection. The aptasensor employs silver-platinum nanoparticles/graphene oxide (AgPt/GO) and Exonuclease III (Exo III) for signal amplification. GO provides high surface area and conductivity for immobilizing AgPt NPs, facilitating the immobilization of aptamer (Apt) probes on the electrode surface. Exo III enzymatically cleaves DNA strands on the electrode surface, releasing DNA segments to amplify the signal further. The synergistic amplification by AgPt/GO and ExoIII enables an extremely wide linear detection range of 0.05 pM-5 nM for Pb2+, with a low detection limit of 0.019 pM. Additionally, the G-quadruplex structure ensures excellent selectivity for Pb2+ detection, resulting in high reproducibility and stability of the aptasensor. The aptasensor was successfully applied to detect spiked Pb2+ in tap water samples, achieving recovery rates ranging from 96 to 108.4 %. By integrating nanomaterials, aptamers and enzymatic amplification, the aptasensor facilitates highly sensitive and selective detection of Pb2+, demonstrating potential for practical applications in environmental monitoring.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Exodesoxirribonucleases , Grafite , Chumbo , Nanocompostos , Platina , Prata , Grafite/química , Chumbo/análise , Chumbo/química , Aptâmeros de Nucleotídeos/química , Exodesoxirribonucleases/química , Técnicas Eletroquímicas/métodos , Platina/química , Nanocompostos/química , Prata/química , Técnicas Biossensoriais/métodos , Nanopartículas Metálicas/química , Limite de Detecção , Poluentes Químicos da Água/análise , Água Potável/análise , Eletrodos , Quadruplex G
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