研究目的
To develop a novel dual-sensor for simultaneous detection of nucleic acids and small chemical molecules using aptamers and photosensitive molecules to regulate DNA hybridization/melting for gene expression control.
研究成果
The developed dual-sensor effectively detects and quantifies nucleic acids and chemical molecules simultaneously with high sensitivity and efficiency, leveraging DNA nanotechnology and photosensitive regulation. It offers advantages in clinical diagnostics by distinguishing endogenous from contaminant chemicals and can be adapted for various targets and promoters.
研究不足
The sensor requires specific aptamers for targets, may have limited sensitivity for very low concentrations, and relies on UV irradiation for optimal performance, which could be a constraint in some applications. The complexity of DNA design and PTG synthesis might limit scalability.
1：Experimental Design and Method Selection:
The study involves designing a dual-sensor with PTG molecules and DNA bubbles to control gene expression based on target presence. Methods include UV-Vis spectroscopy, DNA thermal denaturation, and kinetic studies of RNA polymerase activity.
2：Sample Selection and Data Sources:
Synthetic oligonucleotides (e.g., Oligo 1, Oligo 2), thrombin as a chemical target, and modified DNAs with PTG molecules are used. Data are derived from experimental measurements of melting temperatures, Km and kcat values, and fluorescence protein concentrations.
3：List of Experimental Equipment and Materials:
UV-Vis spectrophotometer for absorption spectra, thermal denaturation setup for Tm measurements, equipment for kinetic assays (e.g., to measure transcription rates), and materials like Tris buffer, PTG molecules, DNA templates, RNA polymerases, and fluorescent proteins.
4：Experimental Procedures and Operational Workflow:
Synthesis of PTG and modified DNAs, introduction of PTG molecules into DNA unwinding and recognition regions, exposure to visible light or UV irradiation at 365 nm, measurement of DNA melting curves, assessment of RNA polymerase kinetics, and quantification of gene expression via fluorescent protein concentrations.
5：Data Analysis Methods:
Analysis of UV-Vis spectra to confirm PTG isomerization, calculation of melting temperatures from denaturation curves, determination of Km and kcat values using Michaelis-Menten kinetics, and correlation of target concentrations with gene expression levels.
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