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Experimental RNAi

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...

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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

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siRNA-Mimetic Ratiometric pH (sMiRpH) Probes for Improving Cell Delivery and mRNA Knockdown.

Madison R Herling¹, Lizeth Lopez Vazquez¹, Ivan J Dmochowski¹

¹Department of Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.

ACS Chemical Biology

|February 5, 2025

Summary

New RNA probes (sMiRpH-2) accurately measure intracellular pH, predicting siRNA delivery success. These probes enable better differentiation between cellular compartments for improved gene silencing strategies.

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Area of Science:

Biochemistry
Molecular Biology
Cell Biology

Background:

RNA interference (RNAi) is a powerful gene silencing tool.
Accurate monitoring of intracellular pH is crucial for understanding cellular processes and optimizing RNA delivery.
Existing pH probes have limitations in dynamic range and cellular differentiation.

Purpose of the Study:

To develop and characterize novel second-generation siRNA-mimetic ratiometric pH probes (sMiRpH-2).
To evaluate the probes' ability to measure intracellular pH and predict siRNA delivery efficacy.
To assess the probes' utility in differentiating cellular compartments and guiding nonviral vector selection.

Main Methods:

Hybridization of 3'-FAM-labeled and 3'-Cy5-labeled RNA strands to form sMiRpH-2 duplexes.
In vitro characterization using fluorescence intensity and lifetime measurements.
In cellulo validation in HeLa and SK-OV-3 cells using RT-qPCR, western blot, flow cytometry, and live cell confocal microscopy.

Main Results:

sMiRpH-2 probes exhibit pH-dependent fluorescence quenching, with a significant dynamic range in the physiological pH window.
Probes accurately measured intracellular pH and correlated with siRNA knockdown efficacy.
FAM-G π-π stacking interaction was identified as a key quenching mechanism.
sMiRpH-2 successfully predicted transfection agent efficacy in challenging SK-OV-3 cells.

Conclusions:

sMiRpH-2 probes are effective tools for real-time intracellular pH monitoring.
These probes enhance the differentiation between endocytic entrapment and cytoplasmic release.
sMiRpH-2 facilitates rapid assessment and optimization of nonviral siRNA delivery vectors.