Efficient construction of functionalized oligonucleotides and DNA duplexes using cross-coupling reactions of nucleoside triphosphates followed by incorporation by DNA polymerase

A novel efficient methodology of cross-coupling of free (non-protected) halogenated nucleoside triphosphates in water was developed and published. This single-step method enables an expeditious and efficient synthesis of modified dNTPs bearing diverse types of aryl and alkynyl substituents as substrates for DNA polymerases. Incorporation of these dNTPs by DNA polymerases using primer extension method followed by denaturation and separation enables isolation of oligonucleotides bearing several specific modifications. Incorporation by PCR leads to DNA duplexes with high density of modifications. Hence, a combination of aqueous cross-coupling reactions of dNTPs with enzymatic incorporation is a new two-step approach to construction of functionalized DNAs. Oligonucleotides bearing amino acid residues or oligonucleotide probes bearing ferrocene or nitro- or aminophenyl labels for electrochemical detection and bioanalytical applications were prepared in this way.

Significant results 2007-1

Čapek P., Cahová H., Pohl R., Hocek M., Gloeckner C., Marx A.:
An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross-Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR.
Chemistry - A European Journal 13: 6196-6203, 2007.

Brázdilová P., Vrábel M., Pohl R., Pivoňková H., Havran L., Hocek M., Fojta M.:
Ferrocenylethynyl Derivatives of Nucleoside Triphosphates: Synthesis, Incorporation, Electrochemistry and Bioanalytical Applications.
Chemistry - A European Journal 13: 9527-9533, 2007.

Cahová H., Havran L., Brázdilová P., Pivoňková H., Pohl R., Fojta M., Hocek M.:
Aminophenyl- and Nitrophenyl-Labeled Nucleoside Triphosphates: Synthesis, Enzymatic Incorporation, and Electrochemical Detection.
Angewandte Chemie International Edition 47: 2059-2062, 2008.



Two-Dimensional Electrophoretic Analysis of a New Human Cell Line EM-G3 Derived from Breast Cancer Progenitor Cells and Comparison with Normal Mammary Epithelial Cells

Breast cancer is one of the most common cancers. Despite extensive research and advances in treatment, the outcome of patients with breast cancer has still not sufficiently improved. It was assumed that cancer cells evolve from stem/progenitor cells distributed throughout the gland. Recently, a new spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma has been established in the Institute of Molecular Genetics in Prague. Its immunocytochemical expression pattern along with the stable genotype shows that EM-G3 cells might be derived from immortalized breast cancer progenitors. We prepared 2-D electrophoretic maps of EM-G3 cell line and using mass spectrometry we identified the main protein spots in order to find characteristic protein markers in this cell line. We have compared the EM-G3 cell line with 2-DE map of proteins from populations of normal mammary epithelial (NME) cells. We identified proteins, which are differently expressed in the studied cells. This study may provide new valuable tools in the early diagnosis of breast cancer.


Significant results 2007-2

Selicharová I., Smutná K., Šanda M., Ubik K., Matoušková E., Buršíková E., Brožová M., Vydra J., Jiráček J.:
2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells.
Proteomics 7: 1549-1559, 2007.



Big Process - Small Model: Triatomic Ag2O+ + Ethylene

Electrospray ionization of aqueous silver nitrate is used for the preparation of the triatomic Ag2O+ cation in the gas phase. The mass-selected cation is capable of epoxidizing ethylene at room temperature, such that gaseous Ag2O+ cation can be regarded as a minimal model for the silver-mediated epoxidation of ethylene - a technical process performed on large scale. The experimental findings are fully supported by the results of quantum-chemical studies, which suggest the formation of a metallaoxetane as the rate-limiting step for both epoxidation and rearrangement to acetaldehyde, which is followed by combustion.


Significant results 2007-3

Roithová J., Schröder D.:
Gas-phase models for catalysis: Alkane activation and olefin epoxidation by the triatomic cation Ag2O+.
Journal of the American Chemical Society 129: 15311-15318 (2007).