Ratiometric, pH-Sensitive Probe for Monitoring siRNA Delivery

Many RNA delivery strategies require efficient endosomal uptake and release. To monitor this process, we developed a 2 '-OMe RNA-based ratiometric pH probe with a pH-invariant 3 '-Cy5 and 5 '-FAM whose pH sensitivity is enhanced by proximal guanines. The probe, in duplex with a DNA complement, exhibits a 48.9-fold FAM fluorescence enhancement going from pH 4.5 to pH 8.0 and reports on both endosomal entrapment and release when delivered to HeLa cells. In complex with an antisense RNA complement, the probe constitutes an siRNA mimic capable of protein knockdown in HEK293T cells. This illustrates a general approach for measuring the localization and pH microenvironment of any oligonucleotide.

Automated summary

To monitor the process of RNA delivery, we developed a pH probe based on 2'-OMe RNA that exhibits enhanced pH sensitivity by guanine proximal effect. The probe, in duplex with a DNA complement, showed a significant FAM fluorescence enhancement from pH 4.5 to pH 8.0, indicating endosomal entrapment and release when delivered to HeLa cells. Moreover, the probe, in complex with an antisense RNA complement, acted as an siRNA mimic and successfully achieved protein knockdown in HEK293T cells, demonstrating its potential for studying the localization and pH microenvironment of oligonucleotides.