Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.

The role of the sequence 1572‐1651 in the C‐terminal tail of the α1C subunit in run‐down of Ca2+ channels was studied by comparing functional properties of the conventional α1C,77 channel with those of three isoforms carrying alterations in this motif. The pore‐forming α1C subunits were co‐expressed with α2δ and β2a subunits in HEK‐tsA201 cells, a subclone of the human embryonic kidney cell line, and studied by whole‐cell and single‐channel patch‐clamp techniques. Replacement of amino acids 1572‐1651 in α1C,77 with 81 different amino acids leading to α1C,86 significantly altered run‐down behaviour. Run‐down of Ba2+ currents was rapid with α1C,77 channels, but was slow with α1C,86. Transfer of the α1C,86 segments L (amino acids 1572‐1598) or K (amino acids 1595‐1652) into the α1C,77 channel yielded α1C,77L and α1C,77K channels, respectively, the run‐down of which resembled more that of α1C,77. These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of α1C,86 renders Ca2+ channels markedly resistant to run‐down. The protease inhibitor calpastatin added together with ATP was able to reverse the run‐down of α1C,77 channels. Calpastatin expression was demonstrated in the HEK‐tsA cells by Western blot analysis. These results indicate a significant role of the C‐terminal sequence 1572‐1651 of the α1C subunit in run‐down of L‐type Ca2+ channels and suggest this sequence as a target site for a modulatory effect by endogenous calpastatin.

For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5'methyl-cytosines (5'mC) in non-bisulfite-treated DNA has been established. The system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5'mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To minimize autofluorescence 150-microm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5'mC in synthetic oligonucleotides hybridized to microarrays with atto molar analytical sensitivity. Using enzymatic fragmented genomic DNA from myeloid leukemia tumor cell lines differences in the methylation status of gene regulatory sequences for E-cadherin, p15/CDKN2b and p16/CDKN2a were demonstrated. Thus, this novel technique can potentially be used for DNA methylation analysis in various scientific fields.