“Multicolor” Electrochemical Labeling of DNA Hybridization Probes with Osmium Tetroxide Complexes

Abstract

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.

Morphology Depot Calcium in Body Tissues in Experimental Modeling Effects of Weightlessness on Earth

“…Существуют гипотезы об альтернативных путях синтеза макроэргов, содержащих фосфаты и транспорта ионов че-рез мембрану при участии мышьяка и антимо-ната [42]. Тетраоксид осмия является специ-фическим окислителем свободного тимина, что используется в способах полимеразно-цепной реакции в случае изучения генов [29]. Антимонатовый буфер обладает свойствами амфотерности, в кислой среде он демонстри-рует основные свойства, а в щелочной среде -кислотные.…”

Electrochemical nucleic acid-based biosensors: Concepts, terms, and methodology (IUPAC Technical Report)

“…Similar applications have been found by other electroactive groups, such as daunomycin, anthraquinone, thionine, bipyridine chelates of Ru or Os, nitrophenyl or aminophenyl groups, etc. Osmium tetraoxide complexes with nitrogen ligands (Os VIII ,L) [24,25] nucleotides and RNAs. Many of the transition-metal-based electroactive tags are electrochemically "tunable" as their redox potentials can be influenced by choice or derivatization of the ligands [23,24].…”

“…Osmium tetraoxide complexes with nitrogen ligands (Os VIII ,L) [24,25] nucleotides and RNAs. Many of the transition-metal-based electroactive tags are electrochemically "tunable" as their redox potentials can be influenced by choice or derivatization of the ligands [23,24]. In addition, some redox labels coupled to nucleobases (such as Fc) respond to the nucleobase type and/or to incorporation into DNA.…”

“…These may be intrinsic components of the NA (e.g., guanine residues) or introduced labels (e.g., multiple redox-active tags used in the tail-labeling techniques [24]); (b) using labels undergoing multi-electron electrochemistry or electrocatalytic processes providing high electron yields (e.g., Os,L at mercury-based electrodes [3,14]); (c) employing biocatalysis (one molecule of the enzyme used as an NA tag can catalyze conversion of many substrate molecules into a detectable product [5,21,27]); (d) in the nanoparticle-based sensing strategies (by tethering one nanoparticle per RP molecule, a large number of the trace atoms is collected per hybridization event; further signal enhancement can be attained by precipitation of additional tracer amount [28]); (e) multilevel signal amplification has been achieved by application of different kinds of particles (microbeads) or nanoobjects such as CNTs, each carrying many redox marker entities (simple redox labels such as Fc, nanoparticles, or enzymes [28]); and (f) in the mercury electrode-based sensors for DNA strand breaks, amplification of the signal is achieved through extensive surface denaturation of the DNA duplex around the lesion [16].…”

Sequence Specific Electrochemical DNA Detection Based on Solution‐Phase Competitive Hybridization

“…Problems with these "immobilization-based" electrochemical DNA detection methods include that the bonding strength of the surface-linked DNA might not survive the high temperature cycling during polymerase chain reaction (PCR). Furthermore, for immobilization-based electrochemical DNA detection schemes, multiplexing would be a huge challenge, as it would add a lot to complexity of the assay and the accuracy of detection may be affected by interference between the detection of different targets [8][9][10]. With these considerations, novel electrochemical DNA detection methods that do not require probe immobilization appear to be a promising alternative.…”