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Peptide Chips and Nucleotide FISH

In contrast to particle physics studying the fundamentals of nature routinely by systematic analyses of huge amounts of data of particle interactions, life sciences are at the beginning to elucidate the complex machinery in cells by systematic studies of bio-molecular interactions. Arrays with combinatorially designed molecules or computer designed bio-probes are typical tools. Such approaches of "systems biology" are the platform on which biophysics and computer engineering meet for novel interdisciplinary projects.

In a living system the main data storage for cellular functions is the cell nucleus containing the DNA that is hierarchically organized in supra-molecular domains and sub-domains that follow functionally controlled structures and arrangements. Cellular data stored in the nucleus or obtained from outside stimuli are processed in a living system by complex cascades and regulatory cycles of proteins. In order to communicate this data processing to other cells, a cell, like in a showcase, displays complex patterns of specifically selected, small fragments of the amino acid chains (peptides) of all activated proteins on its surface.

In our research we address both levels of data presentation (cell nucleus and cell membrane) in order to better understand how a cellular system is functioning or, in cases of diseases, is dysfunctioning.

For systematic investigations of peptide interactions with antibodies or other cells, arrays are required on which thousands of peptides are arranged with amino acid sequences permuting all different 20 amino acids in a combinatorial manner. So far, most available arrays suffer from their low density of peptides and high manufacturing costs. In a novel approach (cooperation with the group of R. Bischoff, F. Breitling, V. Stadler, DKFZ), we designed and built new high-voltage Complementary Metal Oxide Semiconductor (CMOS) chips on which 10,000 different peptides per square centimeter can be synthesized simultaneously. The present generation of the peptide chip has 16,384 metal pixel electrodes (100 x 100 square micrometers) on its surface. Each pixel contains a memory cell in canonical low-voltage CMOS-technology controlling a high-voltage (30 to 100 V) potential area on the top metal layer. Via an IIC interface each pixel can be computer controlled so that arbitrary electrical field patterns are generated, such that electrically charged micro-particles loaded with a given type of amino acids can be addressed on distinct pixel electrodes. The chip attracts the particles from aerosols provided in specially designed chambers (manufactured by the KIP workshop). Thus, we can spatially address a whole layer of different amino acid particles without any concomitant coupling reaction. Peptide synthesis is initiated at once by melting the whole layer of particles. Chemical reaction and washing steps finish the cycle. Several cycles result in combinatorial synthesis of a peptide array. The combination of "wet chemistry" with microelectronics has become a special challenge in the development. We will rise to this challenge by the construction of an automatic device for computer controlled peptide chemistry on the chip mounted in specially developed operating boxes. In addition, an optical detection system for registration of bio-molecular reactions can be implemented into the chips so that separate read-out systems can be omitted.

So far our experiments have demonstrated the functionality and potential of the chip leading to an improvement of the peptide density by two orders of magnitude. Due to the high flexibility of this CMOS peptide chip we will be able to search for peptides relevant for the recognition e.g. of virus infections, bacterial infections, or cancer diseases. Once having found these peptides, our chip may also be a tool to systematically investigate drugs that should interact with the peptides on the cell membrane and finally influence cellular dysfunction.

Moreover, the chip can be used as a tool to (re)program cellular function by presentation appropriate peptides to living cells which may incorporate them. For this purpose a new surface technology has been found allowing cell growth and cellular movement on the chip.

Since peptide presentation on cell surfaces, for instance in correlation to diseases, can be triggered by gene activation or modification, methods for specific probing of functionally correlated gene structures and organisation are required. We developed a meanwhile patented technique using computer designed oligo-nucleotides as probes that specifically bind to complementary DNA sequences in intact cell nuclei. This technique called COMBO-FISH (COMBinatorial Oligo-nucleotide Fluorescence In Situ Hybridization) is based on screening genome data bases for combinations of short nucleotide sequences that uniquely colocalize at a given gene or genome structure. Such probe sets can be designed and tested against the whole genome by computer search and analysis without time consuming biological experiments. Once having found the optimum set of probes the nucleotides are synthesized and labelled by fluorescent dyes so that the respective genome targets can be further analyzed by high resolution fluorescence microscopy (cooperation with the group of C. Cremer). COMBO-FISH probe sets are available for many gene targets that are markers in tumour diagnostic. Due to the design strategy the probe sets can be focused only on the locus of interest. This will allow an improved diagnostic in terms of exact counting of gene copy number changes or structural modifications. Here, we are going to develop a map of gene positions in nuclei of different cell types. Since these positions appear to be functionally correlated, changes of the positions during tumour genesis are studied in order to obtain additional topologic parameters for diagnostics and tumour treatment control.

Peptide chips, COMBO-FISH images, and in general also other approaches of systems biology are going to produce huge amounts of scientific data. All techniques developed in our group will contribute to high through-put screening of bio-molecular interactions. Such data have to be stored and managed. So again we follow the traces of particle physics: At the Bioquant institute we have built hardware systems and data management architectures (cooperation with the group of R. Schneider) based on components successfully installed in huge CERN experiments.