Exploring the Capabilities of Nucleic Acid Polymerases by Use of Directed Evolution 329

Susanne Brakmann and Marina Schlicke 4.3.2 Directed Evolution of Nucleic Acid Polymerases 330 4.3.3 Practical Approaches to the Directed Evolution of Polymerase Function Selection or Screening 331 4.3.3.1 Selection of Polymerases with Altered Activity and Fidelity 331 4.3.3.2 Screening Polymerase Libraries for Altered Activity 331 4.3.4 Genetic Selection of an Error-prone Variant of Bacteriophage T7 RNA Polymerase 333 4.3.5 Screening for Polymerases with Altered Substrate Tolerance 335 4.3.6...

Oxidations with Immobilized Periodinanes

In recent years hypervalent iodine compounds have experienced extensive investigations yielding many results of practical synthetic importance. Supported iodi-nanes, i.e. iodoso or iodine(III) reagents, have been prepared by several groups, mainly as the bis-acetoxyiodoso derivatives 15-18 or as the respective dihalogeno compounds 19 . Iodoso reagents are employed in the oxidation of hydroquinones and phenols that have been exploited in the formation of spiroketals from a variety of tyrosines....

Introduction

''Alzheimer's disease (AD) is a devastating illness that robs humans of their ability to remember, to think and to understand all the things we cherish most about being human.'' (P.F. Chapman) Alzheimer's disease (AD) is an epidemic neurodegenerative disorder claiming millions of victims per year. The aging of the world population will be accompanied by an even higher toll. According to the World Health Organization the prevalence of Alzheimer's disease is 5.5 above 60 years of age and...

Invivo Data of GnRH Binding Spiegelmers

To increase the affinity and improve the temperature-stability of the anti-GnRH DNA sequence the original in-vitro selection was re-initiated after the first selection stage. The selection procedure was therefore performed at 37 C and with greater stringency. After nine additional cycles of selection and amplification the fraction that bound to D-GnRH was increased to 13 . Enriched molecules of the tenth cycle were cloned and sequenced. Thirty-eight out of 48 sequences contained a clone that...

Genetic Selection of an Errorprone Variant of Bacteriophage T7 RNA Polymerase

Bacteriophage

Studies with several RNA viruses have demonstrated their exceptionally high replication error rates that confer genetic flexibility, while resulting in substantial decreases of infectivity 35, 36 . Enhanced mutagenesis might even result in virus extinction, as predicted from virus entry into an ''error catastrophe'' 37, 38 . We are interested in studying and utilizing the link between error-prone polymerases and virus viability and started our search for an error-prone variant with the RNA...

Nonpolar Nucleobase Surrogates

Many nucleotide analogs have been synthesized, and their action on DNA polymerases has been studied. Most of this work has been summarized in recent excellent reviews 3a,d,f,g . In this short summary we focus on selected examples used recently in endeavors devoted to a understanding DNA polymerase selectivity. Most of this work was undertaken to elucidate the non-covalent interactions that govern the formation of the nucleobase pairs. The impact of interstrand base pair hydrogen bonding on...

Info

Bioorganic Receptors for Amino Acids and Peptides Combining Rational Design with Combinatorial Chemistry Carsten Schmuck, Wolfgang Wienand, and Lars Geiger One major research project in our group is currently the design, synthesis, and evaluation of artificial bioorganic receptors for binding amino acids and small oligopeptides in aqueous solvents. Peptides as a substrate are interesting for two reasons - their biological significance and their chemical structure. First, there are many...

Hemoglobin and Myoglobin

Hemoglobin, the constituent of red blood cells responsible for transport of dioxygen, and myoglobin, present in skeletal muscles to store dioxygen, are probably the most thoroughly studied proteins. Hemoglobins were first found in blood because of their very high concentration 15 g per 100 mL in normal human blood . Until recently it seemed that the oxygen carrier function was so specialized that it appeared to be the only role of these Scheme B.10.1. The active site of hemoglobin and...

Spiegelmers Binding to Small Molecules

The first functional mirror-image oligonucleotides, spiegelmers, described in 1996 were generated to bind to small molecules, i.e. the amino acid arginine and the nucleoside adenosine 18, 19 . For in-vitro selection against adenosine, the target in the enantiomeric configuration was covalently coupled to a matrix. A standard characterized and tested for binding to the mirror-image form of the naturally occurring target. The sequences of identified binders are then synthesized in the...

References

1 See, for example, selected excellent reviews and references cited therein a i. Haq, j. Ladbury, J. Mol. Recognit. 2000, 13, 188-197 b p. b. Dervan, r. w. BUrli, Curr. Opin. Chem. Biol. 1998, 3, 688-693 c w. d. Wilson, Nucleic Acids in Chemistry and Biology, g. m. Blackburn, m. j. Gait, eds., IRL Press, Oxford, UK, 1996, pp. 329-374 d b. h. Geierstanger, d. e. Wemmer, Annu. Rev. Biophys. Biomol. Struct. 1995, 24, 463-493 e Recent Advances in DNA Binding Agents. Symposia in print number 4,...

The NOmediated Reduction of FerrylMb and FerrylHb

It has been suggested that heme-mediated redox reactions contribute to the organ dysfunction and or tissue damage which occur in some pathological states characterized by release of Hb and Mb into the extracellular environment 29 . Autoxidation of oxyHb and oxyMb to their met-forms releases superoxide which dis-mutates to dioxygen and hydrogen peroxide, which in turn can cause tissue damage 30 . In addition, oxidation of these two proteins generates potentially cy-totoxic products such as the...

Basf Ag

Research Fine Chemicals GVF E -A30 67056 Ludwigshafen Germany Ecole Polytechnique F d rale de Lausanne EPFL Institut des Materiaux, Laboratoire des Polym res Batiment MX-D, 1015 Lausanne Switzerland NOXXON Pharma AG Max-Dohrn-Stra e 8-10 10589 Berlin Germany Kekule-Institut fur Organische Chemie und Biochemie der Rheinischen Friedrich-Wilhelms-Universitat Bonn Gerhard-Domagk-Strasse 1 53121 Bonn Germany Max Planck Institute of Molecular Physiology Address Otto-Hahn-Stra e 11 44227 Dortmund...