RESUMO
Artificial DNA circuits represent a versatile yet promising toolbox for in situ monitoring and concomitant regulation of diverse biological events within live cells. Nonetheless, their performance is significantly impeded by the diffusion-dominated slow reaction kinetics and the uncontrollable off-target activation. Herein, a self-localized cascade (SLC) circuit is reported for the robust and efficient microRNA (miRNA) analysis in living cells. The SLC circuit consists of the cell-specific localization module and the analyte-specific signal amplification module. By integrating the reaction probes of these two modules, the complexity of the system is reduced to realize the responsive co-localization of circuitry probes and the simultaneous cascade signal amplification. Taking advantage of the specifically activated, self-localized, and cascade design, the SLC circuit successfully achieves the robust miRNA-21 (miR-21) imaging and the accurate cells differentiation. Moreover, the reverse regulation mechanism is successfully explored between messenger RNA (mRNA) and miRNA through the engineered SLC circuit and further elucidates the underlying signaling pathways between them. Therefore, the SLC circuit provides a powerful tool for the sensitive detection of intracellular biomolecules and the study of the corresponding cell regulatory mechanisms.
RESUMO
Laser-free photodynamic therapy (PDT) is a promising noninvasive therapeutic modality for deep-seated tumor, yet is constrained by low efficiency due to the limited stimulation strategies. Herein, a novel miRNA-responsive laser-free PDT was developed through metal-organic frameworks (MOFs)-mediated chemiluminescence resonance energy transfer (CRET) nanoplatform. The photosensitizer chlorin e6 (Ce6)-loaded MOFs were functionalized with hairpin nucleic acids for sensitive responsiveness of tumor biomarker miRNA through catalytic hairpin assembly (CHA), which enabled the amplified assembly of horseradish peroxidase (HRP)-mimicking hemin/G-quadruplex DNAzyme on MOFs. Simultaneously, the on-MOF assembled DNAzymes efficiently catalyzed chemiluminescence reaction to stimulate adjacent Ce6 in the presence of luminol and H2 O2 , thus allowing the CRET-mediated Ce6 luminescence and reactive oxygen species (ROS) generation for self-illuminating PDT. The CRET nanoplatform achieved significant malignant cell apoptosis and tumor inhibition effects without external laser irradiation. It is envisioned that the miRNA-amplified CRET nanoplatform might be a selective and highly efficient antitumor nanomedicine for precise theranostic.
Assuntos
DNA Catalítico , Estruturas Metalorgânicas , MicroRNAs , Neoplasias , Fotoquimioterapia , Porfirinas , Humanos , Luminescência , Transferência de Energia , Fármacos Fotossensibilizantes/farmacologia , Neoplasias/tratamento farmacológico , Linhagem Celular Tumoral , Porfirinas/farmacologiaRESUMO
Cascaded signal amplification technologies play an important role in the sensitive detection of lowly expressed biomarkers of interests yet are constrained by severe background interference and low cellular accessibility. Herein, we constructed a metal-organic framework-encapsulating dual-signal cascaded nucleic acid sensor for precise intracellular miRNA imaging. ZIF-8 nanoparticles load and deliver FAM-labeled upstream catalytic hairpin assembly (CHA) and Cy5-modified downstream hybridization chain reaction (HCR) hairpin reactants to tumor cells, enabling visualization of the target-initiated signal amplification process for double-insurance detection of analytes. The pH-responsive ZIF-8 nanoparticles effectively protect DNA hairpins from degradation and allow the release of them in the acid tumor microenvironment. Then, intracellular target miRNAs orderly trigger cascaded nucleic acid signal amplification reaction, of which the exact progress is investigated through the analysis of the fluorescence recovering process of FAM and Cy5. In addition, DNA@ZIF-8 nanoparticles improve measurement accuracy by dual-signal colocalization imaging, effectively avoiding nonspecific false-positive signals and enabling in situ imaging of miRNAs in living cells. A dual-signal colocalization strategy allows accurate target detection in living cells, and DNA@ZIF-8 provides a promising intracellular sensing platform for signal amplification and visual monitoring.
Assuntos
Técnicas Biossensoriais , Estruturas Metalorgânicas , MicroRNAs , MicroRNAs/genética , MicroRNAs/análise , DNA/genética , Carbocianinas , Hibridização de Ácido Nucleico , Técnicas Biossensoriais/métodosRESUMO
Chemiluminescence resonance energy transfer (CRET)-based assays have shown great potential in biosensing due to their negligible background autofluorescence, yet are still limited by their low sensitivity and short half-life luminescence. Herein, a multistage CRET-based DNA circuit was constructed with amplified luminescence signals for accurate miRNA detection and fixed reactive oxygen species (ROS) signals for cell imaging. The DNA circuit is designed through an ingenious programmable catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and the use of DNAzyme to realize target-triggered precise regulation of distance between the donor and acceptor for CRET-mediated excitation of photosensitizers. In detail, the analyte catalyzes the hybridization of CHA reactants, which leads to the assembly of multiple HCR-mediated DNAzyme nanowires. Subsequently, DNAzymes catalyze the oxidation of luminol by H2O2, and the adjacent photosensitizer chlorin e6 (Ce6) anchored on the DNA nanostructure is stimulated by the CRET process, resulting in the amplified long-wavelength luminescence and the generation of single oxygen signals through further energy transfer to oxygen. The biomarker miRNA can be detected with great sensitivity by integrating the recognition module into a universal platform. Furthermore, the DNA circuit enables CRET-mediated intracellular miRNA imaging, by detecting singlet oxygen signals through a ROS probe. The significant amplification effect is attributed to the robust multiple recognition of the target and the guaranteed transduction of the CRET signal through programmable engineering of DNA nanostructures. The CRET-based DNA circuit achieves amplified long-wavelength luminescence for accurate miRNA detection with low background and ROS-mediated signal fixation for cell imaging, making it a promising candidate for early diagnosis and theranostics.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , MicroRNAs/química , Luminescência , DNA Catalítico/química , Peróxido de Hidrogênio/química , Espécies Reativas de Oxigênio , DNA/genética , Transferência de Energia , Hibridização de Ácido Nucleico , Técnicas Biossensoriais/métodosRESUMO
DNAzyme-based chemiluminescence assay exhibits excellent performance in bioanalysis but their operation in acid conditions remains challengeable. Herein, we constructed an acid-improved DNAzyme-based isothermal enzyme-free concatenated DNA circuit with significantly reduced background and simultaneously improved signal-to-noise ratio for miRNA detection. The chemiluminescence miRNA assay is composed of catalyzed hairpin assembly (CHA), hybridization chain reaction (HCR), and hemin/G-quadruplex DNAzyme units. The analyte initiates the self-assembly of CHA hairpins into numerous dsDNA, which triggers the subsequent autonomous cross-opening of HCR hairpins to generate long nanowires consisting of the hemin/G-quadruplex DNAzyme. The DNAzyme catalyzes the oxidation of luminol by hydrogen peroxide for the cascaded amplified chemiluminescence signal. The acid-improved property was demonstrated to be closely associated with the low catalytic activity of aggregated hemin under acidic conditions and the remained multiple amplified signal through concatenated DNA circuit. The general DNA circuit exhibited high sensitivity for miRNA-21 detection and chemiluminescence imaging under acidic conditions with a recognition hairpin. The acid-improved DNAzyme-based concatenated DNA circuit is promising to expand the application of chemiluminescence assay and provide a valuable strategy for early diagnosis and prognosis of cancer.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , MicroRNAs , Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , DNA Concatenado , Hemina , Luminescência , MicroRNAs/análise , MicroRNAs/genéticaRESUMO
Intrinsic tumor microenvironment (TME)-related therapeutic resistance and nontumor-specific imaging have limited the application of imaging-guided cancer therapy. Herein, a TME-responsive MnO2-based nanoplatform coupled with turn-on and always-on fluorescence probes was designed through a facile biomineralization method for imaging-guided photodynamic/chemodynamic/photothermal therapy (PDT/CDT/PTT). After the tumor-targeting delivery of the AuNCs@MnO2-ICG@AS1411 (AMIT) nanoplatform via aptamer AS1411, the TME-responsive dissociation of MnO2 generated sufficient O2 and Mn2+ with the consumption of GSH for improving PDT efficacy and Fenton-like reaction-mediated CDT. Simultaneously, the released small-sized ICG and AuNCs facilitated PDT and PTT efficacy via the deep tumor penetration. Moreover, the turn-on fluorescence of AuNCs revealed the real-time TME-responsive MnO2 degradation process, and the always-on ICG fluorescence enabled the in situ monitoring of the payload distribution in vitro and in vivo. The AMIT NPs also provided magnetic resonance and thermal imaging guidance for the enhanced PDT, CDT, and PTT. Therefore, this all-in-one nanosystem provides a simple and versatile strategy for multiple imaging-guided theranostic applications.