Direct Voltammetric Analysis of DNA Modified with Enzymatically Incorporated 7-Deazapurines

摘要

Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadeninen(A*) as analogues of natural purine basesnincapable of forming Hoogsteen base pairs, which preventsnthem from being involved in DNA triplexes andntetraplexes. Reduced propensity of the G*- and/or A*-nmodified DNA to form alternative DNA structures isnutilized, for example, in PCR amplification of guanine-richnsequences. Both G* and A* exhibit significantly lowernpotentials of their oxidation, compared to the respectivennatural nucleobases. At carbon electrodes, A* yields annoxidation peak which is by about 200-250 mV lessnpositive than the peak due to adenine, but coincides withnoxidation peak produced by natural guanine residues. Onnthe other hand, oxidation signal of G* occurs at a potentialnby about 300 mV less positive than the peak due tonguanine, being well separated from electrochemical signalsnof any natural DNA component. We show thatnenzymatic incorporation of G* and A* can easily benmonitored by simple ex situ voltammetric analysis of thenmodified DNA at carbon electrodes. Particularly G* isnshown as an attractive electroactive marker for DNA,nefficiently incorporable by PCR. While densely G*-modifiednDNA fragments exhibit strong quenching of fluorescencenof SYBR dyes, commonly used as fluorescentnindicators in both gel staining and real time PCR applications,nthe electrochemical detection provides G*-specificnsignal suitable for the quantitation of the amplified DNAnas well as for the determination of the DNA modificationnextent. Determination of DNA amplicons based on thenmeasurement of peak G*ox is not affected by signalsnproduced by residual oligonucleotide primers or primaryntemplates containing natural purines.