Abstract
Microarrays that mediate the uptake of small molecules into living cells are described. Tissue culture cells were seeded onto glass substrates functionalized locally with fluorescently labelled test substances. In order to enable a localized transferof substances after contact of cells with the substrate, substances were immobilized on the surface either by non-covalent interactions or chemolabile linker groups. These chemolabile linker groups were incorporated into covalently immobilized compounds. Different ester linkages were evaluated as chemolabile linker groups. As model compounds, esters of the carboxy group of a cysteine with the hydroxy groups of carboxyfluorescein-labelled serine amide and tyrosine amide residues or the thiol group of another fluorescein-labelled cysteine amide were generated. Covalent immobilization occurred on maleimide-functionalized glass cover slips. The surface functionalization and release kinetics were assessed by confocal laser scanning microscopy. The fastest release was obtained for the phenolic tyrosine ester. Alternatively, fluorescently labelled peptides were immobilized by non-covalent interactions on glass and on a hydrogel matrix. In order to increase the efficiency of cellular uptake, peptides were N-terminally extended with a cell-penetrating peptide. Uptake of these peptides into cells was confined to the functionalized spots, and was specificfor peptides extended with the cell-penetrating peptide.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ramsay, G., DNA chips- State-of-the-art, Nature Biotech., 16 (1998) 40-44.
Frank, R., High-density synthetic peptide microarrays: Emerging tools for functional genomics and proteomics, Combinatorial Chemistry and High Throughput Screening, 5 (2002) 429-440.
Reimer, U., Reineke, U. and Schneider-Mergener, J., Peptide arrays: From macro to micro, Curr. Opin. Biotech., 13 (2002) 315-320.
Kuruvilla, F. G., Shamji, A. F., Sternson, S. M., Hergenrother, P. J. and Schreiber S. L., Dissecting glucose signalling with diversityoriented synthesis and small molecule microarrays, Nature, 416 (2002) 653-657.
Oldenburg, K. R., Zhang, J.-H., Chen, T., Maffia III, A., Blom, K. F., Combs, A. P. and Chung, T. D. Y., Assay miniaturization for ulta-high throughput screening of combinatorial and discrete compound libraries: A 9600-well (0.2 microliter) assay system, J. Biomol. Screen., 3 (1998) 55-62.
Falsey, J. R., Renil, M., Park, S., Li, S. J. and Lam, K. S., Peptide and small molecule microarray for high throughput cell adhesion and functional assays, Bioconjugate Chem., 12 (2001) 346-353.
Schwenk, J. M., Stoll, D., Templin, M. F. and Joos, T. O., Cell microarrays: An emerging technology for the characterization of antibodies, BioTechniques, 33 (2003) S54-S61.
Ziauddin, J. and Sabatini, D. M., Microarrays of cells expressing defined cDNAs, Nature, 411 (2001) 107-110.
Tsien, R. Y., The green fluorescent protein, Annu. Rev. Biochem., 67 (1998) 509-544.
Derossi, D., Joliot, A. H., Chassaing, G., Prochiantz, A., The third helix of Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem., 269(1994) 10444-10450.
Fischer, R., Mader, O., Jung, G. and Brock, R., Extending the applicability of carboxyfluorescein in solid phase synthesis, Bioconjugate Chem., 14 (2003) 653-660.
Mattingly, P. G., Preparation of 5-and 6-(aminomethyl)fluorescein, Bioconjugate Chem., 3 (1992) 430-431.
MacBeath, G., Koehler, A. N. and Schreiber, S. L., Printing small molecules as microarrays and detecting protein- Ligand interactions en masse, J. Am. Chem. Soc., 121 (1999) 7967-7968.
Hosaka, Y., Yasuda, Y., Seriburi, O., Moran, M. G. and Fukai, K., In vitro secondary generation of cytotoxic T lymphocytes in mice with mumps virus and their mumps-specific cytotoxicity among paramyxoviruses, J. Virol., 57 (1986) 1113-1118.
Barnes-Seeman, D., Park, S. B., Koehler, A. N. and Schreiber S. L., Expanding the functional group compatibility of small-molecule microarrays: Discovery of novel calmodulin ligands, Angew. Chem. Int. Ed., 115 (2003) 2478-2481.
Hergenrother, P. J., Depew, K. M. and Schreiber, S. L., Smallmolecule microarrays: Covalent attachment and screening of alcoholcontaining small molecules on glass slides, J. Am. Chem. Soc., 2000 (2000) 7849-7850.
Kusnezow, W., Jacob, A., Walijew, A., Diehl, F. and Hoheisel, J. D., Antibody microarrays: An evaluation of production parameters, Proteomics, 3 (2003) 254-264.
Bonina, F. P., Arenare, L., Palagiano, F., Saija, A., Nava, F., Trombetta, D. and De Caprariis, P., Synthesis, stability, and pharmacological evaluation of Nipecotic acid prodrugs, J. Pharm. Sci., 88 (1999) 561.
Yang, C., Gao, H. and Mitra, A. K., Chemical stability, enzymatic hydrolysis, and nasal uptake of amino acid ester prodrugs of Acyclovir, J. Pharm. Sci., 90 (2001) 617-624.
Rötzschke, O., Falk, K., Stevanović, S., Jung, G., Walden, P. and Rammensee, H.-G., Exact prediction of a natural T cell epitope, Eur. J. Immunol., 21 (1991) 2891-2894.
Hoff, A., André, T., Schäffer, T. E., Jung, G., Wiesmüller, K.-H. and Brock, R., Lipo-conjugates for the non-covalent generation of microarrays in biochemical and cellular assays, ChemBioChem, 3 (2002) 1183-1191.
Fischer, R., Waizenegger, T., Köhler, K. and Brock, R., A quantitatitive validation of fluorophore-labelled cell-permeable peptide conjugates: Fluorophore and cargo dependence of import, Biochim. Biophys. Acta-Biomembranes, 1564 (2002) 365-374.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hoff, A., André, T., Fischer, R. et al. Chemolabile cellular microarrays for screening small molecules and peptides. Mol Divers 8, 311–320 (2004). https://doi.org/10.1023/B:MODI.0000036241.85469.85
Issue Date:
DOI: https://doi.org/10.1023/B:MODI.0000036241.85469.85