Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.

Osmium tetroxide complexes with nitrogen ligands [Os(VIII)L] have been widely applied as probes of the DNA structure and as electroactive labels of DNA. Here we describe the electrochemical behavior of Os(VIII)2,2-bipyridine (Os, bipy)-base-labeled nucleosides. We show that electroactive label can be introduced also in the nucleoside ribose residues using six-valent osmium complex. Cyclic voltammograms of sugar-Os(VI)-modified ribosides are similar but not identical to those of the base-modified ribosides. Our results showing the electroactivity of sugar modified ribosides pave the way to facile end-labeling of RNA.

We show that polysaccharides (PSs, such as dextran and mannan) can be chemically modified by Os(VI) complexes, yielding electroactive adducts. Os(VI) complexes with different ligands (e.g., temed and 2,2'-bipyridine) produced at pyrolytic graphite electrodes redox couples at different potentials suitable for "multicolor" labeling of PSs and for studies of ligand exchange kinetics. PS-Os(VI)L adducts can be determined not only in their purified forms but also in the reaction mixtures. Dedicated to Professor Karel Vytřas on the Occasion of His 65th BirthdayCarbohydrates belong to the most abundant classes of organic molecules in the biosphere, playing extensive roles in all forms of life. They are one of the main sources of energy for the organotrophic organisms and serve as energy stores (starch, glycogen, inulin) [1]. Many proteins produced by mammalian cells occur as glycoproteins. Recently it has been shown [2,3] that the sugar chains of glycoproteins play important roles in cellular recognition and alterations of the sugar chain structures of tumor glycoproteins can be involved in abnormal social behaviors in cancer cells.Miscellaneous methods have been applied in the analysis of polysaccharides (PSs), including chromatographic [4,5] and colorimetric methods [6] as well as various types of mass spectrometry and nuclear magnetic resonance [7, 8]. On the other hand reports on electrochemical analysis of PSs are scarce. For various reasons chemical modification of carbohydrates proved beneficial. For example, labeling PS molecules by binding of color or fluorescent tags to the reducing end by means of reductive amination have been used [9]. Labeling of nucleic acids [10] using osmium tetroxide complexes with nitrogenous ligands (L) have been widely applied [11]. Os(VIII)L complexes were shown to be excellent probes of the DNA structure [12,13] as well as electroactive labels applicable in DNA sensors [11]. Recently it has been shown that these complexes can be used also as electroactive labels of proteins and probes of their structures [14 -16]. Very recently we have shown that Os(VI)L complexes bind covalently to ribose residues in ribosides and the resulting electroactive conjugates can be sensitively detected at mercury and carbon electrodes [17,18]. These results prompted us to investigate modification of PSs with Os(VI)L complexes and to test electroactivity of the resulting adducts. The modification of saccharides with Os(VI)L is based on the condensation reaction with diol group [19]. Os(VI)L react with acyclic 1,2-diols (glycols) and cyclic cis-1,2-diols. They do mostly not react with trans-1,2-diols, with exception of trans-1,2-cyclohexanediol, trans-1,2-cycloheptanediol [19] and glucose derivatives [20]. The reactions of Os(VI)L with monosaccharides, cyclodextrins and amylose were described [20]. In spite of potential usefulness of these reactions, the literature on modification of carbohydrates with Os(VI)L reagents is scarce and to our knowledge no reports on electrochemical analysis of modi...