A dumbell probe-mediated rolling circle amplification strategy for highly sensitive transcription factor detection

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Transcription factor (TF) detection is crucial for proteome, genome research, and clinical diagnostics.
• Existing methods for TF detection often lack sensitivity, are expensive, or require complex procedures.

Purpose of the Study:

• To develop a novel, ultrasensitive, quantitative, and inexpensive fluorescent-amplified strategy for TF detection.
• To demonstrate the utility of this assay for specific TF detection using TATA-binding protein (TBP) as a model.

Main Methods:

• A hairpin DNA probe with a TF binding site, a primer DNA probe, and a dumbbell probe were utilized.
• Target TF binding inhibits primer hybridization, enabling primer-dumbbell hybridization, ligation, and rolling circle amplification (RCA).
• Quantification was achieved via fluorescent intensity using SYBR Green I (SG).

Main Results:

• The assay specifically detected TATA-binding protein (TBP) with a low detection limit of 40.7 fM.
• A linear detection range from 100 fM to 1 nM was established for TBP.
• The assay requires no modified DNA probes and is performed in a single tube, ensuring simplicity and low cost.

Conclusions:

• The proposed fluorescent-amplified strategy enables ultrasensitive and quantitative TF detection.
• This simple, low-cost, and specific assay has significant potential for bioanalysis and clinical diagnostics.