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S Brakmann

Publications and source records attributed to S Brakmann.

6 recordsLinked to original sources
PubMed2005

Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function.

Polynucleotide polymerases play a crucial role in transmitting genetic information from generation to generation, and they are the most important reagents in biotechnology. Although classical crystal structure analyses as well as biochemical studies have significantly contributed to our understanding of how DNA polymerases function, surprising new insights regarding the importance of certain residues and protein motifs, or of their mutability have been achieved in recent years by evolutionary approaches. Directed evolution has also facilitated the generation of polymerases with tailored substrate repertoires or with stabilities and activities beyond those of their naturally evolved counterparts. Recent new insights in polymerase structure-function relationships and new achievements in the development of tailored polymerases for current methods of nucleic acid synthesis will be summarized in this article.

Directed Molecular Evolution↗
PubMed2001

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

Biochemistry↗
PubMed2001

An error-prone T7 RNA polymerase mutant generated by directed evolution.

Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents. In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population. A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes. We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected. This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection.

Bacteriophage T7↗
PubMed1997

On the generation of information as motive power for molecular evolution.

Molecular evolution can be described as a learning process during which previously inanimate matter developed the ability to organize all the reaction pathways that establish a living system. Common to all natural self-organizing procedures is the ability of matter to store, process and evaluate the information achieved by learning. Genetic information which is stored in RNA or DNA is the object of natural evolution. With the recognition of nature's concepts, evolutionary optimization was applied to biopolymers that are not optimally adapted for particular technical or medical purposes. Information can also be stored in molecules with structures and chemical properties that are completely different from nucleic acids. Therefore, optimization processes that mimic the natural evolutionary strategies can also be applied to small organic molecules. Much effort has been made theoretically and practically to find a certain optimized species within the (hyper)astronomical number of possible sequence alternatives. From a series of computer experiments it can be concluded that it is not necessary to search the entire sequence space in order to find a particular structure; this is advantageous because the diversity of mutant libraries that can realistically be achieved in the laboratory never extends to the number of theoretically possible sequences. Molecular mutant libraries that serve as starting populations for in vitro selection have been constructed for nucleic acids, proteins, peptides and small organic molecules.

Journal Article↗