Quantitative Measurement of Cytosolic and Nuclear Penetration of Oligonucleotide Therapeutics

Abstract

A major obstacle in the development of effective oligonucleotide therapeutics is a lack of understanding about their cytosolic and nuclear penetration. To address this problem, we have applied the chloroalkane penetration assay (CAPA) to oligonucleotide therapeutics. CAPA was used to quantitate cytosolic delivery of antisense oligonucleotides (ASOs) and siRNAs and to explore the effects of a wide variety of commonly used chemical modifications and their patterning. We evaluated potential artifacts by exploring the effects of serum, comparing activity data and CAPA data, and assessing the impact of the chloroalkane tag and its linker chemistry. We also used viral transduction to expand CAPA to the nuclear compartment in epithelial and neuronal cell lines. Using this enhanced method, we measured a 48-h time course of nuclear penetration for a panel of chemically diverse modified RNAs. Moving forward, CAPA will be a useful tool for deconvoluting the complex processes of endosomal uptake, escape into the cytosol, and subcellular trafficking of oligonucleotide therapeutics in therapeutically relevant cell types.

Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy

“…This result helps to rule out degradation artifacts and supports the CAPA data report on the internalization of intact peptides. 45,46 We found that, with the exception of MP3-meta, all stapled peptides had significantly improved cytosolic penetration compared to K1 (Figure 5b). Removing negative charges had modest effects, improving cytosolic penetration by 2−3 fold for some but not all stapled peptides.…”

Investigation of Carboxylic Acid Isosteres and Prodrugs for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme**

“…Here, we report a substantial structure-activity relationship (SAR) study of a series of inhibitors, containing isosteres of the carboxylic acid moiety that is important for ligand affinity to the SIRT5 active site, which produced inhibitors that were equipotent or slightly more potent than the parent compound. Cell penetration was then assessed by applying the chloroalkane penetration assay (CAPA) [49][50][51][52] and target engagement was evaluated by cellular thermal shift assays, revealing similar behavior of the carboxylic acid-containing parent compound and its most potent new analogs. Thus, we show that masking of the tetrazole moiety of the most potent compound can provide improved target engagement in cells to furnish a cellularly active tool compound.…”

Compartment-Specific Measurement of Small Molecule Accumulation into Diderm Bacteria

“…Given that CAPA previously showed low background labeling of mammalian cells in the absence of HaloTag expression, we expected the same when those cells are infected with bacteria. 40,[64][65][66] The principal reason to establish the feasibility of BaCAPA inside mammalian cells is that a large fraction of serious human pathogens (e.g.,…”

“…Given the CAPA previously showed low background labeling of mammalian cells in the absence of HaloTag expression, the same should be expected when those cells are infected with bacteria. 23,[40][41][42] The principal reason to establish the feasibility of BaCAPA inside mammalian cells is that a large fraction of serious human pathogens (e.g., Mycobacterium tuberculosis, Salmonella enterica, Chlamydia trachomatis, and Neisseria gonorrhea) can survive (sometimes exclusively) inside host cells. 43 Moreover, extracellular bacteria can also temporarily reside inside host cells to avoid the action of antibiotics and to promote tissue dissemination.…”