An amphipathic trans-acting phosphorothioate RNA element delivers an uncharged phosphorodiamidate morpholino sequence in mdx mouse myotubes

Abstract

An efficient method for the delivery of uncharged polyA-tailed phosphorodiamidate morpholino sequences (PMO) in mammalian cells consists of employing a synthetic 8-mer amphipathic trans-acting poly-2′-O-methyluridylic thiophosphate triester element (2′-OMeUtaPS) as a transfection reagent. Unlike the dTtaPS DNA-based element, this RNA element is potent at delivering polyA-tailed PMO sequences to HeLa pLuc 705 cells or to myotube muscle cells. However, much like dTtaPS, the 2′-OMeUtaPS-mediated internalization of PMO sequences occurs through an energy-dependent mechanism; macropinocytosis appears to be the predominant endocytic pathway used for cellular uptake. The transfected PMO sequences induce alternate splicing of either the pre-mRNA encoding luciferase in HeLa pLuc 705 cells or the excision of exon 23 from the pre-mRNA encoding dystrophin in myotube muscle cells of the mdx mouse model of muscular dystrophy with an efficiency comparable to that of commercial cationic lipid reagents but without detrimental cytotoxicity.

Synthesis, Characterization, and Function of an RNA‐Based Transfection Reagent

“…However, when 2 -OMeUtaPS is used for the delivery of each sequence in HeLa pLuc 705 cells, uptake of PMO sequence 10 (Fig. 4.81.4) is more efficient (8-10 fold) than that of PNA sequence 9 (Jain et al, 2017), as substantiated by luciferase production measurements (Fig. 4.81.7).…”

“…Furthermore, PMO/CPP conjugates are sensitive to peptidases (Youngblood, Hatlevig, Hassinger, Iversen, & Moulton, ) and produce undesirable toxicity (Wu et al., ). With the objective of improving the delivery of uncharged nucleic‐acid sequences in live mammalian cells, amphipathic, trans ‐acting polythymidylic (dTtaPS) and poly‐2′‐ O ‐methyluridylic (2′‐OMeUtaPS) thiophosphate triester elements have recently been implemented (Jain, Verthelyi, & Beaucage, , ) for efficient delivery of polyA‐tailed PNA and PMO sequences in HeLa pLuc 705 and mdx mouse myotube cells.…”

“…Recognition of the polyA tail of PMO sequence 10 by either dTtaPS or 2′‐OMeUtaPS is required for cellular uptake in mammalian cells (Jain et al., , 2016, ). This can be demonstrated by replacing an A residue with a T residue in the polyA stretch of PMO sequence 10 , which results in a statistically significant decrease in luciferase activity, consistent with weaker binding of 2′‐OMeUtaPS to the modified polyA stretch of PMO sequence 11 (Fig.…”

“…The chemical synthesis of 2 -OMeUtaPS, an octameric phosphorothioate 2 -O-methyluridylic acid functionalized with three N,N-dimethylaminopropyl and four octyl thiophosphate triester functionalities (Fig. 4.81.1), has recently been performed and has been reported to be efficient at delivering polyA-tailed PMO sequences in both HeLa pLuc 705 cells and myotube muscle cells (Jain, Boehler, Verthelyi, Nagaraju, & Beaucage, 2017).…”

“…With the intent of providing novice experimenters with detailed information on work recently published in the scientific literature (Jain et al, 2017), figures and textual materials from that work have been converted into the stepwise procedures delineated herein. This not only facilitates experimental reproduction of the original work but also accounts for unavoidable similarities.…”