Amphipathic trans-acting phosphorothioate DNA elements mediate the delivery of uncharged nucleic acid sequences in mammalian cells

Jain, Harsh V.; Verthelyi, Daniela; Beaucage, Serge L.

doi:10.1039/c5ra12038a

Cited by 10 publications

(23 citation statements)

References 32 publications

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“…12,13 While the cellular internalization of polyA-tailed PMO sequences 5 and 6 (Table 1) exceeded 60% of the total number of cells in three out of the four cell lines investigated, the cellular uptake of PMO sequence 7, lacking the polyA tail, occurred in only 10–15% of the total number of cells in all cell lines under study. 12,13 When assayed in HeLa pLuc 705 cells, the dTtaPS-assisted delivery of the PNA sequence 9 was found more efficient (8–10-fold) than that of PMO sequence 10 on the basis of luciferase activity production (Fig. 3A).…”

Section: Resultsmentioning

confidence: 91%

“…2) were prepared as reported earlier. 12,13 Treatment of commercially available 5′- O -(4,4′-dimethoxytriyl)-2′- O -methyluridine with the phosphordiamidite 1 or 2 and 1 H -tetrazole in a molar ratio of 1: 2 :1, respectively, in anhydrous MeCN afforded the ribonucleoside phosphoramidite 3 or 4 , in yields of ca . 80–85% after purification by chromatography on silica gel and subsequent lyophilization from dry benzene.…”

Section: Resultsmentioning

confidence: 99%

“…The recognition of polyA-tailed PNA or PMO sequences by 2′-OMeUtaPS is specific (Fig. 7) and likely to occur through weak base-pairing interactions; recognition of the polyA tail of these uncharged sequences is strongly inhibited in the presence of urea, 12,13 which is a potent denaturant (Fig. 8).…”

Section: Discussionmentioning

confidence: 99%

“…In this context, we have recently reported the use of a chemically synthesized amphipathic trans -acting poly-thymidylic thiophosphate triester DNA element (dTtaPS) for the delivery of polyA-tailed PNA or PMO sequences in several mammalian cell lines. 12,13 The recognition of the polyA-tailed PNA or PMO sequences by dTtaPS was found to be specific, necessary and sufficient for internalization of PNA and PMO sequences in HeLa pLuc 705 cells; restoration of luciferase activity in this cell line confirmed the correct splicing of luciferase pre-mRNA transcripts. The dTtaPS-mediated cellular internalization of PNA sequences was ~10-fold greater than that of PMO sequences.…”

Section: Introductionmentioning

confidence: 97%

“…The dTtaPS-mediated cellular internalization of PNA sequences was ~10-fold greater than that of PMO sequences. 12 Such a discrepancy in cellular internalization was tentatively rationalized on the basis of a presumably greater affinity of dTtaPS for polyA-tailed PNA than for polyA-tailed PMO sequences. Thus, the development of a trans -acting nucleic acid element that would exhibit a higher affinity for polyA-tailed PMO sequences became a matter of urgency.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2017

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An Amphipathic trans‐Acting Phosphorothioate DNA Element Delivers Uncharged PNA and PMO Nucleic Acid Sequences in Mammalian Cells

Jain

Beaucage

2016

CP Nucleic Acid Chemistry

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An innovative approach to the delivery of uncharged peptide nucleic acids (PNA) and phosphorodiamidate morpholino (PMO) oligomers in mammalian cells is described and consists of extending the sequence of those oligomers with a short PNA-polyA or PMO-polyA tail. Recognition of the polyA-tailed PNA or PMO oligomers by an amphipathic trans-acting polythymidylic thiophosphate triester element (dTtaPS) results in efficient internalization of those oligomers in several cell lines. Our findings indicate that cellular uptake of the oligomers occurs through an energy-dependent mechanism and macropinocytosis appears to be the predo-minant endocytic pathway used for internalization. The functionality of the internalized oligomers is demonstrated by alternate splicing of the pre-mRNA encoding luciferase in HeLa pLuc 705 cells. Amphipathic phosphorothioate DNA elements may represent a unique class of cellular transporters for robust delivery of uncharged nucleic acid sequences in live mammalian cells.

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Trans‐acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high‐throughput microbioreactor system

Hsu

Jain

Williams

et al. 2017

Biotech and App Biochem

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A rapid and cost-effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans-acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High-throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS to deliver a plasmid encoding immunoglobulin IgG1 in a fed-batch serum-free suspension CHO cell culture; dTtaPS -mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid-based transfection reagents under similar conditions. The ability of dTtaPS to deliver plasmids is essentially unaffected by the presence of a silicone-based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS -plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS for transient transfection of plasmids into adherent cells and fed-batch serum-free suspension CHO cells and rapid screening of reagents in a microbioreactor system.

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Amphipathic trans-acting phosphorothioate DNA elements mediate the delivery of uncharged nucleic acid sequences in mammalian cells

Abstract: Amphipathic trans-acting phosphorothioate DNA elements deliver PNA and PMO oligomers in mammalian cells.

Cited by 10 publications

References 32 publications

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

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

An Amphipathic trans‐Acting Phosphorothioate DNA Element Delivers Uncharged PNA and PMO Nucleic Acid Sequences in Mammalian Cells

Trans‐acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high‐throughput microbioreactor system

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