Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells

Crowley, Kathleen S.; Phillion, Dennis P.; Woodard, Scott S.; Schweitzer, Barbara A.; Singh, Megh; Shabany, Hossein; Burnette, Brian L.; Hippenmeyer, Paul J.; Heitmeier, Monique R.; Bashkin, James K.

doi:10.1016/s0960-894x(03)00152-5

Cited by 47 publications

(43 citation statements)

References 14 publications

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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.…”

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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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“…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.…”

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

Hsu

Dervan

2008

Bioorganic & Medicinal Chemistry Letters

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“…Originally, this approach was aimed at creating synthetic molecules 25 for therapeutic gene expression regulation. However, conjugations of these polyamides to 26 fluorescent dyes were developed and shown to be effective in binding double stranded DNA 137 27 and in intracellular localization 138 In summary, conjugates of hairpin pyrrole-imidazole polyamides and fluorophores have found 37 applications as sequence-specific nuclear stains. Due to their gene regulatory properties in 38 which interest was shown as a therapeutic 144 , these minor-groove binders can aid in the study 39 of the mechanism of these therapeutics.…”

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confidence: 99%

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

2015

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7Live-cell imaging has provided the life sciences with insights into the cell biology and 8 dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In 9 this review, we focus on the current fluorescent labeling strategies for nucleic acids, and in 10 particular messenger RNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad 11 range of scientific fields. By giving a background of the available techniques and an evaluation 12 of the pros and cons, we try to supply scientists with all the information needed to come to an 13 informed choice of nucleic acid labeling strategy aimed at their particular needs. 14

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“…[4][5][6] These polyamides have been shown to interfere with protein-DNA interactions, and have the potential to modulate gene expression by designing to recognize specific DNA sequences. 5) For polyamides to be effective in gene regulation, they must reach their target DNA inside the cell.…”

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confidence: 99%

“…[1][2][3] Synthetic polyamides consisting of N-methylpyrrole (Py) and Nmethylimidazole (Im) have received special attention due to their high DNA affinity, sequence specificity, and relatively small size. [4][5][6] These polyamides have been shown to interfere with protein-DNA interactions, and have the potential to modulate gene expression by designing to recognize specific DNA sequences. 5) For polyamides to be effective in gene regulation, they must reach their target DNA inside the cell.…”

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Synthesis and Cellular Effect of a Novel Conjugate of Polyamide and Phospholipid

Liu

Deng

Zheng

et al. 2006

CHEMICAL & PHARMACEUTICAL BULLETIN

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Small molecules with the ability to target predetermined sequences of DNA would be valuable tools in molecular biology and potentially in human medicine.1-3) Synthetic polyamides consisting of N-methylpyrrole (Py) and Nmethylimidazole (Im) have received special attention due to their high DNA affinity, sequence specificity, and relatively small size. [4][5][6] These polyamides have been shown to interfere with protein-DNA interactions, and have the potential to modulate gene expression by designing to recognize specific DNA sequences. 5) For polyamides to be effective in gene regulation, they must reach their target DNA inside the cell. 5,7)However, there are few examples and only circumstantial evidence that suggest that polyamides bind to nuclear DNA targets in living eukaryotic cells. [7][8][9][10][11][12] In the present studies, we designed a conjugate in which a phosphatidylcholine (PC) was attached to the polyamide. The introduction of the phospholipid was expected to improve the permeability of the synthetic molecule through the cell membrane, guide the movement toward to nucleus in cells, and thereby reduce the cytotoxicity. The phospholipid is a primary component of biologicall membrane, and has proven to be noncytotoxic. In particular, the hydrophobic tail of the lipid is possible to greatly benefit the movement of the conjugate toward to nucleus in cells, and subsequently promote the interaction with nucleic DNA. 13) Results and DiscussionPolyamide 10 (O 2 N-PyPy-b-PyPy-b-OEt) and the targeted conjugate 13 (O 2 N-PyPy-b-PyPy-b-PC) were synthesized as shown in Chart 1. b-Alanine (b) functioned as a spacer both in polyamide 10 and conjugate 13. 10 is a regular structure among the synthetic polyamides.14,15) Based on the DNAbinding study results of natural product distamycin and netropsin 16,17) and the pairing rules of polyamides binding to DNA derived by Dervan 14) the polyamide 10 and the polyamide moiety in the designed conjugate 13 were presumed to have an ability of DNA-binding in A/T and T/A pairs rich region.Primarily, DCC coupling method was used in the synthesis.18) Direct coupling of b-alanine with pyrrole monomer Nmethyl-4-nitro-2-trichloroacetylpyrrole 1 without any coupling reagent generated monomer derivative 4 and 5, from which dimeric pyrrole amide 6 and 8 were prepared, respectively. These two dimers were among the key building blocks in the present synthesis. In the preliminary experiment of tetrameric amide synthesis, coupling of acid 7 of the dimer with the amino dimer generated from the hydrogenation of 6 was presumed to give the methyl ester of the tetrameric amide, which would be the desired polyamide and would also serve as another key building block for the conjugate. However, hydrogenation of the nitro group of 6 by the catalysis of palladium on carbon formed many side-products rather than the desired one detected by TLC method. While ethyl ester 8, instead of 6, prepared by the same method as that for the preparation of 6 was used in the reaction the desired reduction product...

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Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells

Cited by 47 publications

References 14 publications

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

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

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

Synthesis and Cellular Effect of a Novel Conjugate of Polyamide and Phospholipid

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