Division of Functional Genome Analysis

Research at the division aims at the development and immediate application of technologies for the production and processing of molecular information at a global cellular level. The overall objectives are an analysis, assessment and description of the realisation of cellular function from genetic information as well as the understanding of the regulation of the relevant processes.

Concerning the analysis of human material, we are establishing systems for early diagnosis, prognosis and an evaluation of the success of disease treatment with a strong accentuation on cancer. Particular attention is paid to pancreatic cancer. Studies are under way, for instance, on the epigenetic modulation of the genome, in combination with transcription factor binding assays, measurements of transcript levels at both mRNA and microRNA level, and the actual protein expression, the last performed mostly by means of complex antibody microarrays. Also, quantitative measurements of protein interactions are pursued at a comprehensive scale.
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One area of our efforts is still work on DNA-, protein- and peptide-microarrays. Technical issues as well as matters of data analysis are being addressed in an attempt to understand the underlying procedural aspects, thereby eventually establishing superior analysis procedures. A more recent field of interest is the pursuit of processes for single molecule detection. The methods are immediately put to use toward an understanding of biological functions and their cellular consequences. Genomic mapping and de novo sequencing have basically ceased as an activity. Second generation high-throughput sequencing, however, is being used as part of functional studies.
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Another line of work aims at a combination of technical advances and access to global biological information toward an in vitro implementation of complex biochemical processes. Motivation is their utilisation in synthetic biology activities for the production of molecules and the establishment of artificial molecular systems. Cell-free biosynthetic production will become important for many biotechnological and pharmacochemical challenges ahead. Complex experimental systems, on the other hand, are meant to complement current systems biology. By means of such in vitro systems, biological models can be evaluated experimentally. Similar to physics, insight into cellular functioning will be gained by an iterative processing of information by experimental and theoretical systems biology. Eventually, this may lead to the establishment of a fully synthetic self-replicating system and - in the long run - an archetypical model of a cell.
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Many projects are pursued in national and international collaborations and programmes. Apart from publications in scientific journals, the division filed a large number of patents/patent applications, of which several have been licensed out or are being utilised in ongoing collaborations with commercial partners.

DNA / RNA Technologies

With the deciphering of the basic sequence information on a genomic scale being completed for very many organisms and with sequencing technology entering a second (or actually third) phase, experimental procedures for an elucidation of the cellular effects and functional consequences of the encoded information have become critical. During about two decades, array technology has established itself as one important methodology for the performance of many such assays at the level of nucleic acids. A few applications are meanwhile done in a routine manner, other even may become obsolete because of better (sequencing) techniques.

The basic arrangement of the microarray format has many facettes beyond the usually reported layout. It can be adapted to serve as a tool in a large variety of applications. New schemes and concepts of utilising microarray technology are still being developed today. Based on our continuous interest in this technology since its early stages, new procedures and formats for the analysis of many biological or biomedically relevant processes are worked at.

One focus of our work are applications that require the ability to produce arrays with double-stranded DNA-probes, for example. Another aspect is the provision of a versatile and therefore widely if not even universally applicable array format, which would be needed particularly for the varying but at the same time highly demanding applications in areas such as routine diagnostics in clinics. Really quantitative assays coupled with a sensitivity level of few individual molecules are other issues that are being worked at. Overall, simplification of the entire processing is still required for reproducible and continuously competitive use of microarrays.

However, also techniques beyond a microarray format are dealt with. Especially experimental formats that aim at studying biological effects at protein level but can technically be reversed to the level of nucleic acids are worked with, since overall the handling of nucleic acids is significantly easier and better to control than dealing with proteins. In addition, particular processes such as in vitro amplification are not available for proteins.

Hoheisel, J.D. (2006) Nature Rev. Genet. 7, 200-210.

Hoheisel, J.D. (2006) Bioanalytik, Elsevier, Heidelberg, 967-978.

Hoheisel, J.D. (2006) Encyclop. Genet. Genom. Prot. Bioinf., Wiley-VHC.

Determination of the sequence of the human genome and knowledge of the genetic code through which mRNA is translated have allowed rapid progress in identification of mammalian proteins. However, less is known about the molecular mechanisms that control expression of human genes and about the variations in gene expression that underlie many pathological states, including cancer. This is caused in part by lack of information about the 'second genetic code' - binding specificities of transcription factors.

Deciphering this regulatory code is critical for cancer research, as too little is known about the mechanisms by which the known genetic defects induce the transcriptional programs that control cell proliferation, survival and angiogenesis. In addition, changes in binding of transcription factors caused by single nucleotide polymorphisms (SNPs) are likely to be a major factor in many quantitative trait conditions, including familial predisposition to cancer.

We aim at developing novel genomics tools and methods for determination of transcription factor binding specificity. These tools are used for identification of regulatory SNPs that predispose to colorectal cancer, and for the characterisation of downstream target genes that are common to multiple oncogenic transcription factors.

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Helwa & Hoheisel (2010) Anal. Bioanal. Chem., in press.

We are pursuing the establishment of a microarray platform that is not assaying one specific biological issue at a time – such as transcription profiles, genotypes or protein-DNA interactions, respectively – but would allow a simultaneous analysis of all these and other aspects in a single experiment. Such an array would allow combining different kinds of molecular markers for a more accurate and informative diagnosis. Especially for analyses on samples of limited quantity, simultaneous assaying might become important. For many assays, it would also be advantageous to perform the actual reaction in homogenous solution rather than on a solid support, since the presence of a surface influences many reactions negatively.

A universal ZIP-code microarray is one option to such ends. This type of microarrays contains a set of unique and distinct (ZIP-code) oligonucleotides that should not have any complementary sequence in any organism and are made solely for the purpose of addressing with a complementary oligonucleotide a particular location on a microarray. The oligonucleotides should have similar thermodynamic properties so that hybridisation can be performed at one experimental condition with identical stringency. Instead of having to produce many different microarrays, a single design can be used for a variety of assays. The actual analysis is carried out with a mixture of probe or primer molecules in homogenous solution. Each oligonucleotide of the mixture is composed of an assay-specific sequence portion that is linked to a distinct, ZIP-code complementary tag-sequence. Only subsequent to the analysis-reaction, the molecules are physically separated by hybridisation to the ZIP-code microarray and therefore made available to individual signal scoring. All probe molecules could assay the same kind of information, such as transcript levels for example, or different types of analysis could be combined.

However, the aspect of avoiding tag-sequences that exhibit similarity to any genome is difficult to achieve. Worse, even very short sequence homologies already lead to some cross-hybridisation and thus a sequence-dependent accumulation of background signal, if complex samples are hybridised. The use of L-DNA could solve this problem. L-DNA is the perfect mirror-image form of the naturally occurring D-conformation of DNA. Therefore, L-DNA duplexes have the same physical characteristics in terms of solubility, duplex stability and selectivity as D-DNA but form a left-helical double-helix. Because of its chiral difference, L-DNA does not bind to its naturally occurring D-DNA counterpart, however. For all the differences, L-DNA is nevertheless chemically fully compatible with the D-form of DNA, so that chimeric molecules can be synthesised. We take advantage of the characteristics of L-DNA toward the establishment of a universal microarray that permits the analysis of different kinds of molecular diagnostic information in a single experiment on a single platform, in various combinations. Also in terms of stability in an impure environment, L-DNA microarrays could be superior, since L-DNA is no substrate for any known enzymatic degradation. The microarrays could therefore be positioned in a fluidic system through which there is a continuous flow of biological material. Apart from handling advantages, more molecules could be captured in a prolonged incubation in a continuous flow system, thus accumulating signal. This could be important for analysing fermentation or production processes, for example, and fits well with currently ongoing developments toward small-scale lab-on-chip devices.

For analyses aiming at a systematic understanding of the processes involved in cellular functioning at the levels of DNA, RNA and protein, we also utilise L-DNA for experimentation in the field of synthetic biology.

Important aspects in microarray analyses are the sensitivity and selectivity of the binding of the assayed DNA molecules. Since the studied DNA-samples usually require a (PCR) amplification and (fluorescence) labelling prior to analysis, time-consuming and costly preparative steps are required, which also might introduce experimental biases. The structural difference between PNA – used as probe on the array – and a DNA-target permits a direct detection of the nucleic acid by mass spectrometry, a process that is much more sensitive than current detection techniques. Thereby, all the preparative steps could be avoided. Upon hybridisation of a DNA or RNA sample to a PNA-array, the phosphates of the nucleid acids can be utilised as an intrinsic label for detection by secondary ion mass spectrometry (SIMS); PNA molecules are lacking phosphate groups entirely. In collaboration with Heinrich Arlinghaus (University of Münster), we have established the processes for analysing genomic DNA directly without the need for amplification or labelling.

Apart from technical developments with respect to synthesis and detection, also the hybridisation behaviour of DNA samples to PNA arrays is being investigated for a precise understanding of PNA-DNA interactions on solid support. The techniques have a wide range of potential applications such as parallel preparation of very many PNA- (or peptide) oligomers for any subsequent use or sequence optimisation of PNA molecules for antisense strategies.

For the production of PNA-arrays, a technique of synthesising PNA oligomers in relatively small quantities but large numbers was established. PNAs are synthesised by an automated process in filter-bottom microtiter plates. The resulting molecules are released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which have been chemically introduced during synthesis. Thus, only full-length PNA-oligomers are attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, do not bind. Different surface chemistries and fitting modifications of the PNA terminus were established. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Based on the results and experience obtained from the synthesis of PNA oligomers, the synthesis and application of complex peptide arrays is performed.

Last partial update: January 2010.
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Functional Tumour Analyses	Proteomics	DNA / RNA Technologies	How to Find Us
- Pancreatic Cancer	- Antibody Microarrays	- Transcription Factor Binding	Open Positions
- Breast Cancer	- Protein Expression Profiling	- shRNA Analysis
- Ovarian Cancer	- Protein Microarrays	- Label-Free Detection / PNA	Group Members
- Other Tumour Entities	- Interaction studies	- Universal Microarrays / L-DNA	- A Typical Day ...
	- Peptide Synthesis	Transcriptional Profiling
Epigenetics		- MicroRNA in Blood and Tissue	Publications / Patents
- NGFN SMP Epigenetics	Single Molecule Detection		Courses & Workshops
- Identification of Drug Resistance		Computational Proteomics (B071)
- Tumour Analyses	Synthetic Biology	Molecular Biophysics	Archive


					Functional Genome Analysis (B070) Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580 D-69120 Heidelberg, Germany.