Simple and Sensitive Method to Assay STAT3 mRNA Triggered by the Self-Assembly of a Three-Way Junction Structure Signal Amplification System.

Obesity is a major global health concern that substantially increases the risk of metabolic disorders. Signal Transducer and Activator of Transcription 3 (STAT3) plays a pivotal role in the progression of obesity by promoting the whitening of brown adipose tissue. In this study, a novel and highly sensitive strategy for STAT3 mRNA detection was developed by integrating three-way junction (3WJ)-mediated strand displacement amplification (SDA) with rolling circle amplification (RCA). The 3WJ structure enables specific target recognition through a proximity effect, enabling accurate discrimination of STAT3 mRNA and triggering primary signal amplification via SDA. The resulting SDA products serve as primers to initiate RCA, achieving secondary signal amplification while eliminating the need for exogenous primers and effectively reducing background signals. This method enables femtomolar (fM)-level detection of target mRNA and exhibits excellent selectivity and stability in complex biological matrices. Overall, the proposed strategy provides a reliable analytical platform for sensitive STAT3 mRNA analysis and broadens its potential applications in obesity research.