Cellular uptake of N-methylpyrrole/N-methylimidazole polyamide-dye conjugates

Belitsky, Jason M.

doi:10.1016/s0968-0896(02)00204-3

Cited by 127 publications

(70 citation statements)

References 13 publications

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“…Fluorescein-labeled polyamide distributed sufficiently in the kidney without any delivery systems and localized in the nuclei of cultured cells for long periods of time. Best et al (35,36) reported that fluorescein-polyamide conjugates exhibit good nuclear uptake in a wide variety of cell lines. In our in vivo experiments, polyamide administered intravenously was delivered sufficiently to the kidney and aorta and was localized to nuclei.…”

Section: Discussionmentioning

confidence: 99%

Development of Gene Silencing Pyrrole-Imidazole Polyamide Targeting the TGF-β1 Promoter for Treatment of Progressive Renal Diseases

Matsuda¹,

Fukuda²,

Ueno³

et al. 2006

Journal of the American Society of Nephrology

102

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Pyrrole-imidazole (Py-Im) polyamides are nuclease-resistant novel compounds that inhibit gene expression by binding to the minor groove of DNA. A Py-Im polyamide that targets rat TGF-␤1 was designed as a gene-silencing agent for progressive renal diseases, and the distribution and the effects of this polyamide on renal injury were examined in Dahl-salt sensitive (Dahl-S) rats. For identification of transcription factor binding elements for activation of the rat TGF-␤1 gene, recombinant TGF-␤1 reporter plasmids were transfected into HEK-293 cells, and promoter activity was measured. Py-Im polyamide was designed to the activator protein-1 binding site of the rat TGF-␤1 promoter. This Py-Im polyamide showed strong, fast, and specific binding to the target DNA in gel mobility shift and Biacore assays. Py-Im polyamide significantly inhibited TGF-␤1 promoter activity and expression of TGF-␤1 mRNA and protein in rat mesangial cells. Intravenously administered fluorescein-labeled polyamide distributed to the kidney of rats. Py-Im polyamide significantly inhibited expression of TGF-␤1 mRNA and protein in the renal cortex of Dahl-S rats and reduced the increase in urinary protein and albumin in Dahl-S rats independent of changes in blood pressure. These results indicate that Py-Im polyamide that targets TGF-␤1 will be a novel gene-silencing agent for the TGF-␤1-associated diseases, including progressive renal diseases.

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Section: Discussionmentioning

confidence: 99%

Development of Gene Silencing Pyrrole-Imidazole Polyamide Targeting the TGF-β1 Promoter for Treatment of Progressive Renal Diseases

Matsuda¹,

Fukuda²,

Ueno³

et al. 2006

Journal of the American Society of Nephrology

102

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“…Internalization was found to be energy-dependent, and the internalized peptide was observed in the endocytic vesicles of living cells (30). Dervan, Johansson, and their coworkers have reported similar cell-fixation artifacts for the transduction of N-methylpyrrole/Nmethylimidazole polyamides (31) and PTD-green fluorescent protein fusions (32).…”

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confidence: 84%

Pathway for Polyarginine Entry into Mammalian Cells

2004

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Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R 9 ), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R 9 is found in the cytosol as well as the nucleolus when cells are fixed, this peptide is observed only in the endocytic vesicles of live cells. Co-localization studies with vesicular markers confirm that PTDs are internalized by endocytosis rather than by crossing the plasma membrane. The inability of R 9 to enter living cells deficient in heparan sulfate (HS) suggests that binding to HS is necessary for PTD internalization. This finding is consistent with the high affinity of R 9 for heparin (K d = 109 nM). Finally, R 9 is shown to promote the leakage of liposomes, but only at high peptide:lipid ratios. These and other data indicate that the PTD-mediated delivery of molecules into live mammalian cells involves: (1) binding to cell-surface HS, (2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles. Facilitating the delivery of molecules into mammalian cells is of substantial interest (1-6).Developing the means to do so can generate both insights into cellular function and potential applications in biomedicine. As early as 1965, Ryser noted that mixing polylysine or other cationic macromolecules with proteins greatly increases the uptake of those proteins by cells (7). In 1988, Frankel and Green independently discovered that the RNA-binding HIV 1 TAT protein was capable of crossing lipid bilayers (8,9). The dissection of HIV TAT function has revealed that a small cationic domain, residues 47-57, is responsible for transduction (10). Other cationic, nucleic acid-binding sequences are likewise capable of entering cells (11,12). Peptide-like molecules and even non-peptidyl polycations are capable of crossing lipid bilayers (11,13). These cationic "protein transduction domains" (PTDs) are not only capable of entering cells but also can be used to deliver molecular cargo (14-16), such as proteins, oligonucleotides, and small molecules (1,(17)(18)(19)(20).What are the physicochemical characteristics of cationic PTDs? A natural backbone is not necessary for transduction, as D-amino acid (21), peptoid (18), oligocarbamate (22), β-peptide (23,24), and 6-aminocaproic acid (25) analogs are capable of entering cells. In contrast, the positive charge of cationic PTD is essential for transduction, and arginine is preferred over † This work was supported by National Institutes of Health Grant GM44783. 1 Abbreviations: CHO, Chinese hamster ovary; CS, condroitin sulfate; GAG, glycosaminoglycan; HIV, human immunodeficiency virus; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; PC, phosphatidyl choline; PTD, protein transduction domain; R 9 , nonaarginine; TAT, transactivator of transcription; TAMRA-R 9 , 5-(and 6-)carboxytetramethylrhodamine conjugat...

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“…[18][19][20][21] In particular, with laser microscopy it is possible to visualize the uptake of polyamidefluorophore conjugates as they traffic unaided to the nucleus of live cells. [3][4][5][6] This advance has helped to usher in an era of gene regulation. 8-11 Traditionally, we have used a qualitative scoring system to rate the extent of nuclear localization in cell culture.…”

mentioning

confidence: 99%

“…The degree of nuclear localization varies across polyamide design, fluorophore selection, and cell type. [3][4][5][6] Quantitating the polyamide concentration within the cell nucleus would be an improvement over the current "yes/no" rating, thus creating a digital readout of cellular uptake.…”

mentioning

confidence: 99%

Quantitating the concentration of Py-Im polyamide–fluorescein conjugates in live cells

Hsu

Dervan

2008

Bioorganic & Medicinal Chemistry Letters

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Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide-fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Upon treatment with polyamide, the concentration in the nucleus of live HeLa cells is calculated to be between 0.1-0.5 μM, which is significantly lower than the 2 μM dosage concentration. In contrast, the observed nuclear concentration in U251 cells is closer to the dosage concentration, indicating a cell line-specific increase in uptake for this class of compounds. Although confocal microscopy and flow cytometry generate disparate values, taken together these experiments suggest that the polyamide concentration inside the cell nucleus is lower than it is outside the cell.Keywords gene regulation; fluorescence; confocal microscopy; flow cytometry Hairpin pyrrole-imidazole polyamides are synthetic ligands that target predetermined DNA sequences with affinities comparable to those of naturally occurring DNA-binding proteins. 1,2 These cell-permeable small molecules have been shown to localize to the nucleus of living cells 3-7 and regulate endogenous gene expression. 8-12 Polyamides selectively bind in the minor groove of DNA according to a set of "pairing rules," where each heterocyclic ring pair targets a specific Watson-Crick base pair. 1,2 The antiparallel pairing of N-methylpyrrole (Py) and N-methylimidazole (Im) aromatic rings targets the C·G base pair, while Im/Py discriminates G·C. 13-16 The Py/Py pair recognizes A·T and T·A, while chlorothiophene can be paired with Py to distinguish T·A at the N-terminus. 17Polyamide-small molecule conjugates have been utilized in a number of applications. 18-21 In particular, with laser microscopy it is possible to visualize the uptake of polyamidefluorophore conjugates as they traffic unaided to the nucleus of live cells. 3-6 This advance has helped to usher in an era of gene regulation. Figure S1), confocal laser scanning microscopy images (Supplementary Figure S2), and experimental details (Supplementary Information).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. scoring system to rate the extent of nuclear localization in cell culture. 5,6 Upon incubation with polyamide and direct imaging of cells, the fluorescence intensity in the nucleus is compared to that in the medium. Positive scores are assigned when the nuclear staining exceeds that of the medium. The degree of nuclear localization varies across p...

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Cellular uptake of N-methylpyrrole/N-methylimidazole polyamide-dye conjugates

Cited by 127 publications

References 13 publications

Development of Gene Silencing Pyrrole-Imidazole Polyamide Targeting the TGF-β1 Promoter for Treatment of Progressive Renal Diseases

Development of Gene Silencing Pyrrole-Imidazole Polyamide Targeting the TGF-β1 Promoter for Treatment of Progressive Renal Diseases

Pathway for Polyarginine Entry into Mammalian Cells

Quantitating the concentration of Py-Im polyamide–fluorescein conjugates in live cells

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