Base and conformational specificity of an amine modification of DNA

We have investigated the site and conformational preference of the reaction of a formaldehyde/amine reagent with DNA. Previous investigations of this laboratory have established that this reagent will react with native DNA, placing a positively charged amine moiety on the duplex that will survive exhaustive dialysis. The resulting adduct is duplex and base stacked in character, possessing B backbone geometry with a higher average winding angle and exhibiting remarkable stability with respect to the A‐form, Z‐form, or the single‐strand denaturated species. In this current investigation, we have found that the stability of the adduct is dramatically reduced if the DNA is converted to mononucleotides, thus obviating the usual approach of nuclease digestion and chromatography for the identification of the modified nucleotides. Using indirect approaches, we have established that the reactive site that survives removal of the equilibrium concentrations of CH 2 O and amine is the exocyclic amino group of the guanine bases. This conclusion is based on (1) the positive correlation between GC content and the extent of of adduct formation under standard reaction conditions (27°C, 0.63 M CH 2 O, 0.007 M n ‐butylamine, pH 7); (2) decreases in the level of substitution of amine in DNA, which has this site blocked by trinitrobenzene modification; and (3) failure of poly(dI‐dC) to retain amine upon dialysis. Raman spectra of the derivatized poly(dG‐dC) show enhanced 2′‐ endo B character, with no marked shifts in the position of any of the lines, indicating the absence of any ring structures involving the N7 and the 06 of G. In standard reaction mixtures, other sites may react but this phenomenon appears to be minimal under conditions that do not favor fluctuational opening of base pairs. In the latter case, excess loading of amine on high GC content polymers produces a CD spectrum that is similar to one produced by poly(dA‐dT) in the “X”‐form [M. Vorlickova, E. Minyat, and J. Kypr (1984) Biopolymers 23 , 1–4]. This conformation is lost, however, upon removal of excess reagents by dialysis and cannot be reestablished, in the absence of unbound amine and formaldehyde. The reaction is specific for the B‐form of polynucleotides as demonstrated by the failure of poly(dG‐m 5 dC) in the stable Z‐form to exhibit substantial reaction. The B‐form of this polymer will react readily with the retention of 0.23 moles amine/mole nucleotide under our standard reaction conditions. Although retaining B‐form geometry, this adduct will lose significant amounts of amine when exposed to conditions that stabilize the Z‐form. This behavior is in contrast to that previously reported for poly(dG‐dC), which retains both the B‐form and all of its amine content under equivalent conditions. The present results reflects the greater Z‐forming potential of the methylated polymer. These observations lead us to the conclusion that the B‐form provides a GC locus that favors site binding of cations in the minor groove and stabilizes B geometry. This specific site is lost in the B → Z conversion. Thus, the cationic interactions that stabilize the Z‐form must occur at alternate loci.