Sensitive and selective detection of microRNA using hyperbranched rolling circle amplification based on symmetric...

Area of Science:

• Molecular Biology
• Biotechnology
• Biochemistry

Background:

• MicroRNA (miRNA) expression changes are crucial for understanding tumor-associated functions and disease biomarker development.
• Detecting low-abundance and highly homologous miRNAs presents significant challenges in molecular diagnostics.

Purpose of the Study:

• To develop a highly sensitive and selective method for detecting specific microRNAs (miRNAs).
• To enable accurate miRNA quantification in complex biological samples without extensive pre-processing.

Main Methods:

• A novel exponential amplification strategy combining a symmetric dumbbell-type probe (SDTP) and toehold-initiated hyperbranched rolling circle amplification (HRCA).
• Utilized toehold-mediated strand displacement for target recognition and SDTP activation, followed by phi29 DNA polymerase-driven HRCA for signal amplification.
• Demonstrated single-base mismatch discrimination and direct detection in cell lysates without RNA extraction.

Main Results:

• Achieved exponential amplification of let-7a, enabling sensitive detection across a wide concentration range (1.00 fmol·L^-1 to 1.00 nmol·L^-1).
• Established a detection limit as low as 214 amol·L^-1 with linear correlation between fluorescence intensity and logarithmic miRNA concentration.
• Successfully distinguished miRNAs with single-base mismatches and validated accuracy in serum samples via spiking recovery experiments.

Conclusions:

• The developed method offers a simple, sensitive, and specific approach for miRNA detection in various biological samples.
• This technique shows significant potential as a valuable tool for miRNA analysis in clinical diagnostics and research.