SIRNA-MIMETIC RATIOMETRIC FLUORESCENT PH PROBES FOR CELLULAR RNA DELIVERY AND SCREENING

Loading...
Thumbnail Image
Degree type
Doctor of Philosophy (PhD)
Graduate group
Chemistry
Discipline
Chemistry
Biochemistry, Biophysics, and Structural Biology
Biology
Subject
Fluorescence
Oligonucleotides
pH sensing
Ratiometry
RNA delivery
siRNA
Funder
Grant number
License
Copyright date
2024
Distributor
Related resources
Author
Herling, Madison, Ross
Contributor
Abstract

First described in C. elegans in 1998, RNAi and the siRNA by which it is triggered have proven to be transformative discoveries for basic, translational, and clinical research applications. Despite 26 years of research progress, only six siRNA drugs have been approved by the FDA for clinical use and they all target liver hepatocytes. The overwhelming consensus among researchers in this field is that many novel nanocarriers with improved targeting, delivery, and endosomal escape efficiency are needed. To monitor endosomal escape, we developed a 2′-OMe RNA-based siRNA-mimetic ratiometric pH (sMiRpH) probe with a pH-invariant 3ʹ-Cy5 and 5ʹ-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 in HeLa cells. In complex with an antisense RNA complement, the probe constitutes an siRNA mimic capable of protein knockdown in HEK293T cells. Second-generation (sMiRpH-2) probes were developed by hybridizing a 3′-FAM-labeled 2′-OMe RNA strand with a 3′-Cy5-labeled 25mer RNA strand. These duplexes demonstrated 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. 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 was found to be predictive of knockdown efficacy. A sMiRpH-2 probe was successfully tested with two transfection agents in more challenging SK-OV-3 cells, which highlights its use for the rapid assessment of non-viral siRNA delivery vectors.

Advisor
Dmochowski, Ivan, J
Date of degree
2024
Date Range for Data Collection (Start Date)
Date Range for Data Collection (End Date)
Digital Object Identifier
Series name and number
Volume number
Issue number
Publisher
Publisher DOI
Journal Issue
Comments
Recommended citation