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Vitamin B₁₂ as a carrier of peptide nucleic acid (PNA) into bacterial cells.

ABSTRACT: Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B₁₂ to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B₁₂ was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B₁₂ conjugates; namely the vitamin B₁₂ azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B₁₂ and PNA were tested, including a disulfide bond. We found that vitamin B₁₂ transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)₃K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B₁₂ as a carrier of PNA oligonucleotides into bacterial cells.

SUBMITTER: Rownicki M

PROVIDER: S-EPMC5550456 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Vitamin B<sub>12</sub> as a carrier of peptide nucleic acid (PNA) into bacterial cells.

Równicki Marcin M Wojciechowska Monika M Wierzba Aleksandra J AJ Czarnecki Jakub J Bartosik Dariusz D Gryko Dorota D Trylska Joanna J

Scientific reports 20170809 1

Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B<sub>12</sub> to transport peptide nucleic acid (PNA) oligomers into ...[more]

PMID: 28794451

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Project description:We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA length (to adjust binding affinity). In general, the carrier CPP-PNA constructs including the ones with decanoyl modification provided significant increase of the activity of unmodified antisense PNA as well as of antisense octaarginine-PNA conjugates. Antisense activity, and by inference cellular delivery, of unmodified antisense PNA was enhanced at least 20-fold at 6 ?M upon the complexation with an equimolar amount of nonamer carrier decanoyl-CPP-PNA (Deca-cPNA1(9)-(D-Arg)8). The antisense activity of a CPP-PNA ((D-Arg)8-asPNA) (at 2 ?M) was improved 6-fold and 8-fold by a heptamer carrier CPP-PNA (cPNA1(7)-(D-Arg)8) and hexamer carrier decanoyl-CPP-PNA (Deca-cPNA1(6)-(D-Arg)8), respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP-PNA carriers may be used as effective cellular delivery vectors for different types of antisense oligomers and also allows use of combinations of (at least two) different CPP ligands.

Dataset Information

Vitamin B₁₂ as a carrier of peptide nucleic acid (PNA) into bacterial cells.

Publications

Vitamin B<sub>12</sub> as a carrier of peptide nucleic acid (PNA) into bacterial cells.

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Dataset Information

Vitamin B12 as a carrier of peptide nucleic acid (PNA) into bacterial cells.

Publications

Vitamin B<sub>12</sub> as a carrier of peptide nucleic acid (PNA) into bacterial cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Vitamin B₁₂ as a carrier of peptide nucleic acid (PNA) into bacterial cells.