Potassium ferrate as a DNA chemical sequencing reagent and probe of secondary structure.

Endogenous iron in aerobic systems has been implicated in causing oxidative damage to nucleic acids and other cellular components [l]. Complex oxygenated ions of iron (IV) and iron (V), possibly formed as transient intermediates in Fenton reactions, have been postulated to contribute to this process [2]. These hypervalent iron species may be generatedty radjolysis of aqueous solutions containing the ferrate ion (FeO4 ) which is a derivative of iron (VI). Ferrate ion is a very powerful oxidant, its standard redox potential increasing markedly with decreasing pH from 0.72 V in alkaline media to 2.2 V in strong acid [3]. Its potassium salt K FeO4, which is readily soluble in water, is a convenient source otferrate ions. Although it is known to be capable of oxidizing certain organic functional groups [4], the action of ferrate ion on nucleic acids does not appear to have been investigated hitherto. In this communication, we report results on the degradation of 2'deoxyribonucleosides by potassium ferrate and its ability to induce alkali-labile cleavage sites in DNA. Potassium ferrate was synthesised by the method of Thompson er al. [5] and recrystallised from 6 M KOH. Its concentration in solution was determined spectrophotometrically [6] from measureynt-s at its visible absorption maximum at 508 nm ( E ~ ~ 1100 Mcm l). In preliminary experiments, the relative reactivity of potassium ferrate towards the four common 2 deoxyribonucleosides deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC) and thymidine (dT) was examined in aqueous solution at pH 8. The individual deoxyribonucleosides (from Sigma Chemical Co.), at a concentration of 2 mM, were mixed with different molar ratios of potassium ferrate and left at mom temperature for 1 h before adding allyl alcohol to quench the reaction. Reversed-phase HPLC, in combination with UV spectrophotometry, was then used to recover and quantify the amount of unreacted starting material in each case. From this, the percentage of each deoxyribonucleoside degraded under the reaction conditions was calculated (and is given in parentheses). For equimolar concentrations of deoxyribonucleoside and potassium ferrate the order of reactivity was dG (30%). dT (20%). dC (14%) and dA (4%). However, when a 1O:l molar ratio of ferrate to deoxyribonucleoside was employed, the extent of degradation was increased and the order was as follows: dG (loo%), dC (62%). dT (25%) and dA (13%). Thus, dG was established as the most readily oxidized deoxyribonucleoside and dA as the most resistant, with dC and dT exhibiting intermediate reactivity. The effect of potassium ferrate oxidation on intact DNA was investigated by means of gel sequencing experiments with endlabelled synthetic oligodeoxyribonucleotides. The oligonucleotides were routinely synthesised on an Applied Biosystems Model 380B i n s u u p t and purified by gel electrophoresis; they were labelled with P at 5'-terminus by the action of T4 polynucleotide kinase and [y PIATP. Standard protocols were used for chemical sequencing of DNA [7]. Typically, a 10 1 1 aliquot of an aqueous solution of the end-labelled oligonucleotide (containing 10-20 pmol) was mixed with 10 pi of a buffered solution of potassium ferrate in an Eppendorf vial. After 2 min at O°C. the reaction was stopped by the addition of 1 1 1 of allyl alcohol. The DNA was recovered by ethanol precipitation and, where appropriate, heated with 1 M piperidine. at 90°C for 30 min. Finally, the DNA samples were loaded and separated on sequencing gels (40 x 18 cm, 0.3 mm thick; containing 16% polyacrylamide and 7 M urea) alongside the products of chemical sequencing reactions. The gel patterns were visualised by autoradiography. Several single stranded end-labelled oligonucleotides were treated with millimolar concentrations of potassium ferrate over the pH range 7.5 to 10. Under these conditions, no fragments produced by direct cleavage of the DNA backbone were evident on the sequencing gel autoradiograms, suggesting that the deoxyribose residues remained intact. However, when the oxidized oligonucleotides were subsequently heated with piperidine to detect alkali-labile cleavage sites, extensive fragmentation occurred. Although the pattern varied somewhat with pH and femte concentration, the most intense cleavage fragments migrated on the gels with the guanine (G) and thymine (T) bands in the chemical sequencing reference lanes. Relatively weak cleavage occurred at the sites of cytosine (C) bases and essentially none was discernible at adenine (A). These results imply that the oxidation of the G and T bases in DNA by ferrate ion produces lesions which are unstable under alkaline conditions and lead to DNA chain scission, most probably through the well established mechanism of base loss followed by p-elimination at the abasic site. By reducing the concentration of ferrate ion to 10 pM, at pH 7.5, the cleavage at T could be suppressed giving a weak G-specific sequencing reaction. The 34-mer 5 -ATCCGATCGGACCGGACTGA'ITGATCG TGATCAT-3' was used in establishing the 'G+T sequencing specificity of potassium ferrate. Substitution of C(26) in this oligonucleotide by 5-methylcytosine led to the appearance of a cleavage band at this position whose intensity was comparable to the bands at G and T sites. Potassium ferrate is therefore able to distinguish between deoxycytidine and 5-methyldeoxycytidine residues in DNA. Weakly acidic potassium permanganate is the only other chemical sequencing reagent which has been reported [8] to share this useful property. Modification experiments with an oligonucleotide adopting a hairpin structure demonstrated that G and T bases in double stranded DNA are less susceptible to oxidation by potassium ferrate than those in single stranded DNA. Thus, the sequencing gel patterns revealed more intense cleavage fragments for G and T bases within the loop region than in the duplex stem. Hence, with further development, potassium ferrate may find application as a chemical probe for delineating DNA secondary structure. In conclusion, potassium ferrate degrades 2'deoxyribonucleosides in the decreasing order dG>dC-dT>dA. Because it generates alkali-labile lesions at guanine and thymine bases in DNA, it can be used as a 'G+T chemical sequencing reagent; it can also discriminate between cytosine and 5methylcytosine. It may be possible to exploit the differential reactivity of potassium ferrate towards bases in single and double stranded regions of DNA in probing local secondary structure.