Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.
| Functional
Genome Analysis (B070) Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580 D-69120 Heidelberg, Germany. |
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Functional
Tumour Analyses |
Proteomics |
DNA / RNA Technologies |
How to find
us |
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- Pancreatic
Cancer |
- Antibody Microarrays |
- Transcription
Factor Binding |
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| - Breast Cancer | - Cancer Profiling |
- shRNA
Analysis |
Open
Positions |
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| - Other Tumour Entities | - Personalised Proteomics |
- PNA against
sncRNAs |
Group Members | |
| - Protein Microarrays |
- Universal
Microarrays / L-DNA |
- A Typical Day … | ||
| Epigenetics (DNA methylation) | - Interaction Studies | |||
| - Correction of Measurement Biases | Transcript Studies | Publications / Patents | ||
| - Identification of Drug Resistance | Single Molecule Detection | - MicroRNA in Blood and Tissue | ||
| - Tumour Analyses | - Functional and Diagnostic Variations | > Computational Proteomics (B071) | ||
| Synthetic Biology | > Chip-Based Peptides | |||
| Compound Analyses | Archive | > Molecular Biophysics |
 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.
.. 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 ceased as an activity. Second generation high-throughput sequencing, however, is being used as part of functional tumour studies. .. 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. .. 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. |
Very early experiment on the production of oligonucleotide microarrays by light-directed in situ synthesis. A pattern resembling letters was repeatedly projected onto a glass surface, triggering oligomer synthesis. Subsequenly, a fluorescently labelled oligonucleotide of complementary sequence was hybridised to this chip, producing the pattern shown. |
With the
deciphering of the
basic sequence information on a genomic scale being completed for very
many
organisms and with sequencing technology having entered 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 (as well as proteins). Several applications are meanwhile done in
a
routine manner, other
may even 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 and partly based on earlier results, new procedures and formats for the analysis of biological or biomedically relevant processes are worked at. One focus of our work are applications that support functional studies, such as the aspect of genome-wide screening for essential genes. Other approaches require the ability to produce arrays with double-stranded DNA-probes, for example for protein binding studies. 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. However, also the development of techniques beyond the microarray format are being pursued. In one project, experimental formats are worked at that aim at studying biological effects at protein level but can technically be reversed to the level of nucleic acids. Since the handling of nucleic acids is significantly easier overall and better to control than dealing with proteins, such procedures could be advantageous. In addition, particular processes such as in vitro amplification are not available for proteins.
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 Decoding
pooled RNAi screens by means of lentiviral shRNA libraries RNA
interference (RNAi) has become a popular and important tool for the
analysis of gene
function.
Loss-of-function studies, commonly performed by transfection of short
interfering RNAs (siRNAs), have greatly facilitated functional analyses
of the
human transcriptome. However, there are major downsides to siRNA
experiments,
most importantly the transient inhibition of gene expression as well
as their
inefficient transfection into non-dividing cells.
... In order to overcome those limitations, short hairpin RNA (shRNA) expression vectors were developed, which can be stably integrated into a target cell's genome via retro- or lentiviral gene transfer. Intracellular processing of shRNAs results in short duplex RNAs with siRNA-like properties. Viral integration ensures not only a broad range of infectable target cell types but also the stable expression of specific shRNAs, resulting in the permanent reduction of the targeted gene product. Recently, complex shRNA expression libraries have become available. Those libraries allow the targeted knockdown of thousands of different genes. ... Using such lentiviral vector shRNA libraries and barcode arrays or sequencing analysis for decoding of the pooled RNAi screens, we are able to quantify precisely the abundance of individual shRNAs. The use of tiling arrays has improved performance even further, particularly in view of really genome-wide studies, clearly outperforming the commonly used analysis via the shRNAs’ half hairpin sequences. However, particularly for large-scale assays with many thousands of shRNA constructs, next-generation sequencing has become the method of choice for read-out. ... We used the technique to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens, for example. In this study, we identified 28 shRNAs to impair fully or partially the viability of the breast carcinoma cells. Further studies are under way, both for the elucidation of basic regulative processes associated to cancer and for the identification of pathways that are affected by particular drugs or compounds. Next to breast cancer, we use the technique also for getting more detailed information on the functional effects of particularly potentially druggable gene products in pancreatic cancer. Böttcher et al. (2010) BMC Genomics 11, 7.  Böttcher& Hoheisel (2010) Curr. Genom. 11, 162-167. |
 sncRNAomics – High throughput comparative sncRNAome
analysis in major Gram-positive human pathogenic bacteria: functional
characterisation by a systems biology approach and peptide nucleic acid
(PNA) drug
design
 In
recent years, small non-coding RNAs (sncRNAs) and especially microRNAs
(miRNAs)
have been identified as key regulators of several cellular processes.
In
bacteria, sncRNAs have attracted considerable attention as an emerging
class of
gene expression regulators. The ERA-Net consortium sncRNAomics intends to utilise
bioinformatics, novel high-throughput sncRNA screening methods,
whole-genome
transcriptomics and proteomics, coupled with existing robust molecular
characterisation methods to provide comprehensive information
regarding production, regulation and pathogenic implications of sncRNAs
in five
major high-risk Gram-positive pathogens.
... This information will be used to design novel potential therapeutics based on sncRNA-complementary peptide nucleic acids (PNAs). PNA production is being done according to newly developed protocols for a low-cost, small-scale, high-throughput PNA-/peptide synthesis. PNAs show major advantages over common nucleic acid-structured therapeutic agents. As they lack the phosphodiester backbone, they are much more stable against enzymatic digestion and in addition display higher binding affinities in hybridising reactions. PNAs designed to bind tightly to target sncRNAs will penetrate the bacterial cell, hybridise to the respective sncRNA and counteract its effect in pathogenicity. In parallel, the knowledge gained in the project will be used to develop a sensitive diagnosis system, which will be able to detect sncRNAs in the fmol range directly at point-of-care in a very short time. ... Mraheil et al. (2010) Microb. Biotechnol. 3, 634-657.  ... ... Based on
several earlier projects on PNA,
a technique was
established
of synthesising and purifying PNA
oligomers in
relatively small quantities but large numbers. PNAs are
synthesised by an automated process in filter-bottom microtiter plates.
The
resulting molecules are released from the solid support and purified by
taking advantage of terminal protection groups. In consequence, only
full-length PNA-oligomers are binding to the purification matrix
whereas
truncated
molecules, produced during synthesis because of incomplete condensation
reactions, do not bind. Different surface chemistries and fitting
modifications
of the PNA terminus have been established and filed for patent protection. Based on the results
and experience
obtained with PNA oligomers, also protocols
for the parallel synthesis
and purification of
peptides
were established.
... The quick synthesis and purification of large numbers of peptides or PNAs is being offered by a spin-off company resulting from this project.
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 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. ... 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. Hoheisel, J.D. (2006) Nature Rev. Genet. 7, 200-210. |
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