Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates

Uffe Koppelhus; Satish Kumar Awasthi; Vladimir Zachar; Henrik Uffe Holst; Peter Ebbesen; Peter E. Nielsen

doi:10.1089/108729002760070795

Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates

Uffe Koppelhus, Satish Kumar Awasthi, Vladimir Zachar, Henrik Uffe Holst, Peter Ebbesen, Peter E. Nielsen

Transcription, RNA, and Gene Medicine Program

Abstract

Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.

Original language	English
Journal	Nucleic Acid Therapeutics
Volume	12
Issue number	2
Pages (from-to)	51-63
Number of pages	13
ISSN	1087-2906
DOIs	https://doi.org/10.1089/108729002760070795
Publication status	Published - Apr 2002

Keywords

Amino Acid Sequence
Animals
Biological Transport, Active
Cell Line
Drug Carriers
Fluorescent Dyes
HeLa Cells
Humans
Maleimides
Microscopy, Confocal
Molecular Sequence Data
Molecular Structure
Peptide Nucleic Acids/chemistry
Peptides/chemistry
Subcellular Fractions/metabolism
U937 Cells

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1089/108729002760070795

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title = "Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates",

abstract = "Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.",

keywords = "Amino Acid Sequence, Animals, Biological Transport, Active, Cell Line, Drug Carriers, Fluorescent Dyes, HeLa Cells, Humans, Maleimides, Microscopy, Confocal, Molecular Sequence Data, Molecular Structure, Peptide Nucleic Acids/chemistry, Peptides/chemistry, Subcellular Fractions/metabolism, U937 Cells",

author = "Uffe Koppelhus and Awasthi, {Satish Kumar} and Vladimir Zachar and Holst, {Henrik Uffe} and Peter Ebbesen and Nielsen, {Peter E.}",

year = "2002",

month = apr,

doi = "10.1089/108729002760070795",

language = "English",

volume = "12",

pages = "51--63",

journal = "Nucleic Acid Therapeutics",

issn = "1087-2906",

publisher = "Mary AnnLiebert, Inc. Publishers",

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TY - JOUR

T1 - Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates

AU - Koppelhus, Uffe

AU - Awasthi, Satish Kumar

AU - Zachar, Vladimir

AU - Holst, Henrik Uffe

AU - Ebbesen, Peter

AU - Nielsen, Peter E.

PY - 2002/4

Y1 - 2002/4

N2 - Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.

AB - Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.

KW - Amino Acid Sequence

KW - Animals

KW - Biological Transport, Active

KW - Cell Line

KW - Drug Carriers

KW - Fluorescent Dyes

KW - HeLa Cells

KW - Humans

KW - Maleimides

KW - Microscopy, Confocal

KW - Molecular Sequence Data

KW - Molecular Structure

KW - Peptide Nucleic Acids/chemistry

KW - Peptides/chemistry

KW - Subcellular Fractions/metabolism

KW - U937 Cells

U2 - 10.1089/108729002760070795

DO - 10.1089/108729002760070795

M3 - Journal article

C2 - 12074365

SN - 1087-2906

VL - 12

SP - 51

EP - 63

JO - Nucleic Acid Therapeutics

JF - Nucleic Acid Therapeutics

IS - 2

ER -

Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates

Abstract

Keywords

UN SDGs

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