siRNA-Mimetic Ratiometric pH (sMiRpH) Probes for ImprovingCell Delivery and mRNA Knockdown

Second-generation siRNA-mimetic ratiometric pH probes (sMiRpH-2) were developed by hybridizing a 3′-FAM-labeled 2′-OMe RNA strand with a 3′-Cy5-labeled 25mer RNA strand. These duplexes demonstrated the silencing of cytoplasmic mRNA targets in HeLa cells as measured by RT-qPCR and supported by western blot analysis. Fluorescence intensity and lifetime measurements revealed that a single guanosine (G) positioned adjacent to FAM achieves substantial static quenching at pH 5, with additional collisional quenching rendering the dye almost nonemissive. A FAM-G π–π stacking interaction was evidenced by a red-shifted absorbance spectrum for FAM. Decreased quenching at near-neutral pH enhances the FAM dynamic range in the physiologic pH window and improves the differentiation in cells between endocytic entrapment and cytoplasmic release. Flow cytometric analysis of intracellular pH and uptake using sMiRpH-2 was corroborated by live cell confocal microscopy and found to be predictive of knockdown efficacy. A sMiRpH-2 probe successfully predicted the relative efficacy of two transfection agents in more challenging SK-OV-3 cells, which highlights its use for the rapid assessment of nonviral siRNA delivery vectors.