Bioassay using a labeled oligonucleotide obtained by in vitro selection

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Biology

Background:

• Developing selective and sensitive bioassays is crucial for detecting specific molecules.
• Oligodeoxyribonucleotide (DNA) aptamers offer high specificity for molecular recognition.
• Fluorescent labeling is a common technique for signal detection in bioassays.

Purpose of the Study:

• To develop a novel bioassay system for the detection of Reactive Green 19 (RG19).
• To evaluate the impact of fluorescein labeling on the structural integrity and binding affinity of a DNA aptamer targeting RG19.
• To establish a semiquantitative method for determining RG19 concentration.

Main Methods:

• In vitro selection was used to obtain a DNA aptamer with high affinity for RG19.
• The DNA aptamer was labeled with fluorescein.
• Circular dichroism spectroscopy was employed to assess conformational changes after labeling.
• Binding affinity of labeled and non-labeled aptamers was compared using RG19.
• A semiquantitative assay was performed using the labeled DNA aptamer and RG19-immobilized gel.

Main Results:

• A DNA aptamer that selectively binds to RG19 was successfully generated.
• Fluorescein labeling did not significantly alter the DNA aptamer's conformation, as confirmed by circular dichroism.
• The binding affinity of the fluorescein-labeled DNA aptamer to RG19 was equivalent to that of the non-labeled aptamer.
• The developed system allowed for the semiquantitative determination of RG19 concentration.

Conclusions:

• Fluorescein labeling is a suitable modification for the RG19-targeting DNA aptamer without compromising its binding characteristics.
• The novel bioassay system utilizing the labeled DNA aptamer provides a reliable method for the semiquantitative analysis of RG19.
• This approach demonstrates the potential of aptamer-based assays for sensitive and specific molecular detection.