LAUSR

MicroRNAs (miRNAs) are associated with various cellular processes and have been recognized as potential biomarkers for many human diseases. The sensitive and accurate determination of miRNA expression levels in biological specimens is highly significant for understanding their biological functions and clinical diagnosis. Mass spectrometry (MS) has shown its potential to study bioactive molecules, however, direct MS analysis of miRNAs is often hampered by limited sensitivity. For sensitive detection of miRNAs, indirect methods are generally employed through the use of DNA probes labeled with peptides or metal elements. In this work, we proposed a novel MS-based label-free strategy for miRNA quantification. A dual-amplification system was developed by using a padlock probe containing the poly(thymine) sequence in combination with rolling circle amplification (RCA). The specific recognition of target miRNA by the padlock probes produced long single-stranded DNAs containing poly (adenine) segments, which guaranteed the specificity of detection and realized primary amplification. Then the RCA products were extracted and treated with acid to release a large number of free adenines as reporter molecules for secondary signal amplification. Overall, the quantification of target miRNA was carried out by signal switching from high-molecular-weight RCA products to highly sensitive small molecule of adenine. The developed method achieved a linear detection range from 200 amol to 100 fmol for miRNA-21 with a limit of detection of 50 amol, and successfully applied to detect endogenous miRNA-21 levels from lung cancer cells. Overall, the present study provides a sensitive, specific MS-based method for miRNA detection and holds great potential for further application of MS technology to detect other biomarkers in biomedical research and early clinical diagnosis.