Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA

Abstract

Toward the development of a universal, sensitive and convenient method of DNA (or RNA) detection, electrochemically active oligonucleotides were prepared by covalent linkage of a ferrocenyl group to the 5'-aminohexyl-terminated synthetic oligonucleotides. Using these electrochemically active probes, we have been able to demonstrate the detection of DNA and RNA at femtomole levels by HPLC equipped with an ordinary electrochemical detector (ECD) [Takenaka,S., Uto,Y., Kondo,H., Ihara,T. and Takagi,M. (1994) Anal. Biochem., 218, 436-443]. Thermodynamic and electrochemical studies of the interaction between the probes and the targets are presented here. The thermodynamics obtained revealed that the conjugation stabilizes the triple-helix complexes by 2-3 kcal mol-1 (1-2 orders increment in binding constant) at 298 K, which corresponds to the effect of elongation of additional several base triplets. The main cause of this thermodynamic stabilization by the conjugation is likely to be the overall conformational change of whole structure of the conjugate rather than the additional local interaction. The redox potential of the probe was independent of the target structure, which is either single- or double stranded. However, the potential is slightly dependent (with a 10-30 mV negative shift on complexation) on the extra sequence in the target, probably because the individual sequence is capable of contacting or interacting with the ferrocenyl group in a slightly different way from each other. This small potential shift itself, however, does not cause any inconvenience on practical applications in detecting the probes by using ECD. These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acids due to the stability of the complexes, high detection sensitivity and wide applicability to the target structures (DNA and RNA; single- and double strands) and the sequences.

Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA

“…They also report that the current flow through the double helix is sensitive even to single-base mismatches, paving the way for the direct electrochemical identification of point mutations (9). In an attempt to reduce high backgrounds arising from the inappropriate binding of hybridization indicators to unhybridized DNA, sandwich-type detectors have also been developed and commercialized (29)(30)(31). Sandwich approaches employ a surface-confined, electrochemically silent probe sequence to bring target DNA strands to the electrode surface.…”

Eletrodos modificados com DNA: uma nova alternativa em eletroanálise

“…Outra possibilidade de determinação de DNA foi proposta por Ihara et al 62 por meio da síntese de composto conjugado de oligonucleotídeo-ferroceno. Neste trabalho o poli(T) 12 -ferrocenila forma complexos, por hibridização, com poli(A) e dsDNA, formando dupla e tripla hélices, respectivamente.…”

Electrochemical and Spectroscopic Investigations of Protonated Ferrocene-DNA Intercalation

“…1 A number of investigations reveal that the most likely target of antitumor drugs is cellular DNA, and the interaction is such that the tertiary structure of DNA is distorted, thereby inhibiting the replication and transcription machinery of the cell. 2,3 This damage may lead to various pathological changes in living organisms.…”