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Updated: Dec 22, 2025
MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as A Novel Detection and Quantification Method
Published on: October 7, 2025
Analysis of microRNA-induced silencing complex-involved microRNA-target recognition by single-molecule fluorescence
1Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong, 518055, China.
Abstract:
MicroRNAs (miRNAs) are important regulators of gene expression that control almost every physiological and pathological process. Although the complementarity between the seed region of a miRNA and its target mRNA is usually deemed as the key determinant in the miRNA-target recognition in animals, the mechanism of their recognition still remains enigmatic as more and more exceptions challenge the seed rule. Herein, we employ single-molecule fluorescence resonance energy transfer (smFRET) to investigate human miRNA-induced silencing complex (miRISC)-involved miRNA-target recognition with either perfect base pairing or poor seed match in real time. Our results demonstrate that the recognition between mammalian miRNA and its target with perfect base pairing proceeds in a two-state model as prokaryotic guide DNA-mediated recognition, suggesting a conserved pattern of guide RNA/DNA strand recognition. In addition to the general rule of miRNA-target recognition, our results reveal that annealing between miRNA and its target with poor seed match proceeds in a stepwise way, which is in accordance with the increase in the number of conformational states of miRNA-target duplex accommodated by the miRISC, suggesting the structural plasticity of human miRISC to conciliate the mismatches in seed region. This new dynamic information revealed by smFRET has an important implication for comprehensive understanding of the role of miRISC in the target recognition in mammals.
Insights
Single-molecule assays reveal how microRNAs (miRNAs) bind targets. Perfect matches use a two-state model, while poor matches show stepwise binding, highlighting miRISC plasticity.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) are crucial gene regulators in physiological and pathological processes.
- The seed region complementarity is traditionally key for miRNA-target recognition, but exceptions challenge this rule.
- The precise mechanism of miRNA-target recognition remains incompletely understood.
Purpose of the Study:
- To investigate miRNA-target recognition within the human miRNA-induced silencing complex (miRISC) in real time.
- To elucidate the dynamics of miRNA-target binding with both perfect and imperfect seed matches.
- To understand the role of miRISC structural plasticity in accommodating target mismatches.
Main Methods:
- Employed single-molecule fluorescence resonance energy transfer (smFRET).
- Studied human miRISC-mediated recognition of miRNA targets.
- Analyzed miRNA-target interactions with perfect base pairing and poor seed matches.
Main Results:
- Perfectly matched miRNA-target recognition follows a conserved two-state model, similar to prokaryotic systems.
- miRNA-target annealing with poor seed matches occurs stepwise.
- The number of conformational states of the miRNA-target duplex within miRISC increases with mismatches, indicating structural plasticity.
Conclusions:
- Mammalian miRNA-target recognition with perfect pairing shares conserved features with prokaryotic systems.
- Human miRISC exhibits structural plasticity to accommodate seed region mismatches during target recognition.
- This study provides dynamic insights into miRISC function in mammalian target recognition.
