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Updated: Jul 20, 2026
Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Published on: April 14, 2015
Self-assembly of protein-DNA hybrids dedicated to an accelerated and self-primed strand displacement amplification
Guoqiao Huang1, Chan Li2, Rong Wu2
1Key Laboratory of Laboratory Medicine, Ministry of Education of China, and Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, PR China; Department of Laboratory Medicine, Jintang First People's Hospital, West China Hospital Sichuan University Jintang Hospital, Chengdu, Sichuan, 610400, PR China.
Background:
High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis.
Results:
Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis.
Significance:
This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.
