Phosphorothioate and 2′-Fluoro Ribose Substitution-Mediated Hydrophobic Effect Empowers RNA Probes for Highly Sensitive and Subtype-Selective miRNA Imaging

MicroRNAs (miRNAs) are challenging to detect due to their sequence similarity, short length, and low abundance. Compared to traditional DNA probes, RNA probes exhibit superior affinity, thereby mitigating low accuracy and poor specificity caused by misrecognition and off-target effects. Nonetheless, the susceptibility to enzymatic degradation severely limits their application in biological systems. To address this, we engineered F-S-RNA probes by integrating phosphorothioate backbones (-P═S) and 2'-fluoro ribose substitution (2'-F substitution). The hydrophobic effect induced by these modifications significantly disrupts the polar salt bridges between F-S-RNA and RNase 1, thereby enhancing the nuclease resistance of the probes. Meanwhile, it strengthens the hydrophobic stacking within the F-S-RNA/miRNA complex, consequently improving the hybridization. The probes achieve a 10-fold lower detection limit (5 pM) than conventional DNA probes (50 pM) and demonstrate exceptional specificity in discriminating let-7 family subtypes. Leveraging these advantages, we investigated the small-molecule (TPEN and LI71) modulation of LIN28-mediated let-7 suppression in situ, uncovering a subtype-selective regulatory pattern. Notably, compared to TPEN, LI71 selectively inhibits LIN28's suppression of let-7b, undetectable with DNA probes. This work establishes a powerful miRNA detection platform while demonstrating the value of chemically modified RNA probes in deciphering post-transcriptional regulation and therapeutic development.