Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Scheme 1. Top: Formulas of Purine Nucleobases (A,<br />
G) and Their 7-Deaza Analogues (A*, G*) a<br />
in primer extension (PEX) experiments. 12,13 Nevertheless, many<br />
of the nucleobase conjugates have displayed less facile incorporation<br />
at adjacent positions 7,9,10 and only some of them have been<br />
efficiently used in PCR. 13,14<br />
The 7-deazapurines, 7-deazaguanine (G*), or 7-deazaadenine<br />
(A*) (Scheme 1) are substitutes of standard purine nucleobases<br />
incorporable into DNA by PCR. Although the rate of G* and A*<br />
incorporation by DNA polymerases has been reported 15 to be<br />
lower, compared to the “parent” purines, both of these nucleobase<br />
analogues allow efficient sequence-specific DNA amplification by<br />
PCR and it has been possible to prepare PCR products with a<br />
high density of the corresponding modification. The 7-deazapurines<br />
are able to form Watson-Crick base pairs, maintaining<br />
pairing specificity of the respective natural nucleobases, but cannot<br />
form Hoogsteen pairs due to absence of the N7 atom (which is<br />
substituted by CH group, Scheme 1). Thus, the 7-deazapurines<br />
cannot be involved in triplex and tetraplex DNA structures. 16<br />
Reduced tendency of the “deaza-modified” DNA to adopting<br />
multistranded conformations has been utilized to improve PCR<br />
amplification of G-rich sequences such as (CGG)n repeat, 17 the<br />
length of which is analyzed during molecular diagnostics of<br />
fragile X syndrome. Similarly, 7-deaza-8-azaguanine has been<br />
used to create G-rich DNA probes with reduced propensity to<br />
aggregate and improved specificity. 18 Absence of the nitrogen<br />
atom at the 7-position in G* was also utilized in a study of<br />
sequence-specificity of DNA modification with cisplatin 19 and<br />
in studies of echinomycin-DNA interaction modes. 20 Moreover,<br />
DNA substituted with G* or A* has been shown to resist<br />
cleavage with certain endonucleases. 15<br />
a<br />
Atoms at 7-position are highlighted in red. Bottom: Watson-Crick<br />
and Hoogsteen base pairing. N7 is involved in the Hoogsteen pairing<br />
of natural purines.<br />
(13) Cahova, H.; Pohl, R.; Bednarova, L.; Novakova, K.; Cvacka, J.; Hocek, M.<br />
Org. Biomol. Chem. 2008, 6, 3657–3660.<br />
(14) Raindlova, V.; Pohl, R.; Sanda, M.; Hocek, M. Angew. Chem., Int. Ed. 2010,<br />
49, 1064–1066.<br />
(15) Seela, F.; Roling, A. Nucleic Acids Res. 1992, 20, 55–61.<br />
(16) Palecek, E. Crit. Rev. Biochem. Mol. Biol. 1991, 26, 151–226.<br />
(17) Cao, J.; Tarleton, J.; Barberio, D.; Davidow, L. S. Mol. Cell. Probes 1994,<br />
8, 177–180.<br />
(18) Kutyavin, I. V.; Lokhov, S. G.; Afonina, I. A.; Dempcy, R.; Gall, A. A.; Gorn,<br />
V. V.; Lukhtanov, E.; Metcalf, M.; Mills, A.; Reed, M. W.; Sanders, S.;<br />
Shishkina, I.; Vermeulen, N. M. J. Nucleic Acids Res. 2002, 30, 4952–4959.<br />
(19) Cairns, M. J.; Murray, V. Biochim. Biophys. Acta 1994, 1218, 315–321.<br />
(20) Sayers, E. W.; Waring, M. J. Biochemistry 1993, 32, 9094–9107.<br />
6808 <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
Table 1. Oligonucleotides Used in This Work a<br />
prim rnd 5′-CATGGGCGGCATGGG-3′<br />
prim noG 5′-TACTCATCATATCAA-3′<br />
temp rnd16 5′-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3′<br />
temp noG 5′-AATATAAATATATTGATATGATGAGTA-3′<br />
p53-for 5′-GAGGTTGTGAGGCGCTGCCC-3′<br />
p53-rev 5′-TCCTCTGTGCGCCGGTCTCT-3′<br />
a The template strands (temp rnd16 , temp noG ) used in PEX experiments<br />
were 5′end-biotinylated.<br />
Compared to the standard purines, 7-deazapurines exhibit<br />
significantly lower potentials of their oxidation. 9,21,22 G* is thus<br />
easily photooxidized by intercalated ethidium and this photooxidation<br />
has been interrogated as a function of distance, nucleotide<br />
sequence and integrity of π-stacking in studies of DNA-mediated<br />
charge transfer. 21 The G* ability of being selectively oxidized by<br />
a redox mediator with relatively low redox potential, such as<br />
Ru(dmb)3 3+/2+ (dmb )4,4′-dimethyl-2,2′-bipyridine), compared<br />
to A* together with natural G requiring stronger oxidants, such<br />
as Ru(bpy)3 3+/2+ (bpy )2,2′-bipyridine), has been utilized in a<br />
PCR-coupled electrochemical technique proposed for parallel<br />
detection of two genes. 22 Mediated electrooxidation of G* (or<br />
G) was also applied in an indirect electrochemical real time<br />
monitoring of PCR via measuring consumption of the respective<br />
dNTPs. 23 To our best knowledge, direct electrochemical<br />
analysis of DNA with incorporated G* or A* as electrochemically<br />
oxidizable tags has not been reported to date.<br />
In this paper we report on adsorptive transfer stripping<br />
voltammetric analysis of DNA with enzymatically incorporated G*<br />
or A* residues at a carbon electrode. We show that, particularly<br />
G* producing a specific signal separated from those yielded by<br />
natural DNA components, can be utilized as an excellent electroactive<br />
label suitable for monitoring of DNA amplification by PCR.<br />
MATERIALS AND METHODS<br />
Material. Synthetic ODNs (Table 1) were purchased from<br />
VBC genomics (Austria). Templates used in experiments involving<br />
the magnetoseparation procedure were biotinylated at their 5′<br />
ends. Plasmid pT77 bearing wild type p53 cDNA insert 24 (used<br />
as primary template for PCR amplification of the 347-bp fragment)<br />
was isolated from E. coli cells using Qiagen Plasmid Purification<br />
Kit and linearized with EcoR I restrictase (Takara). Streptavidincoated<br />
magnetic beads (MBstv) were purchased from Novagen<br />
(Germany), DyNAzyme II DNA Polymerase from Finnzymes<br />
(Finland), Pfu DNA Polymerase from Promega (U.S.), unmodified<br />
nucleoside triphosphates (dATP, dTTP, dCTP and dGTP),<br />
SYBR Green I, SYBR Gold and Stains-All reagent from Sigma,<br />
7-deaza-dGTP and 7-deaza-dATP from Jena Bioscience. Other<br />
chemicals were of analytical grade.<br />
Primer Extension (PEX). The primer (0.7 µM) was mixed<br />
with corresponding template ODN (0.7 µM), dNTPs (100 µM<br />
each; composition of the dNTP is specified in the text and Figure<br />
legends for individual experiments) and the DyNAzyme II DNA<br />
(21) Kelley, S. O.; Barton, J. K. Chem. Biol. 1998, 5, 413–425.<br />
(22) Yang, I. V.; Ropp, P. A.; Thorp, H. H. Anal. Chem. 2002, 74, 347–354.<br />
(23) Defever, T.; Druet, M.; Rochelet-Dequaire, M.; Joannes, M.; Grossiord, C.;<br />
Limoges, B.; Marchal, D. J. Am. Chem. Soc. 2009, 131, 11433–11441.<br />
(24) Hupp, T. R.; Meek, D. W.; Midgley, C. A.; Lane, D. P. Cell 1992, 71, 875–<br />
886.