Inhibition of nonenzymatic depurination of nucleic acids by polycations

Abstract

DNA base depurination is one of the most common forms of DNA damage in vivo and in vitro, and the suppression of depurination is very important for versatile applications of DNA in biotechnology and medicine. In this work, it was shown that the polycations chitosan (Cho) and spermine (Spm) strongly inhibit DNA depurination through the formation of polyion complexes with DNA molecules. The intramolecular electrostatic interaction of positively charged polycations with DNA efficiently suppresses the protonation of purine groups, which is the key step of depurination. Importantly, the optimal pH for Cho's inhibition of depurination is significantly different from that of Spm. Cho is very effective in the inhibition of depurination in highly acidic media (pH: 1.5-3), whereas Spm is found to suppress the chemical reaction near neutral pH, as well as in acidic solutions. This remarkable pH specificity of the two biorelevant polycations is attributed to the difference in the pK_a values of the amino groups. The relevance of our results with the biological roles of biogenic polycations is also discussed.

Keywords: DNA protection; chitosan; depurination; polycations; spermine.

Figure 1

Inhibition by Cho of depurination of N30 at pH 2.0 and 37 °C. (A) Time courses in the presence of Cho (closed rhombuses) and its absence (closed circles); N/P = 3. (B) Effect of the amount of Cho on the first‐order rate constant of depurination. Structure of Cho is presented in the bottom panel.

Figure 2

The inhibition by Cho and glucosamine (Gla) on depurination from dAMP, and the inhibition by glucosamine (Gla), in place of Cho, on the depurination of N30. N/P = 3 at pH 2.0 and 37 °C.

Figure 3

Inhibitory effect of Cho for the depurination of ODNs of various sequences. (A) First‐order rate constants for the release of each of adenine and guanine; (B) the corresponding inhibitory ratios. N/P = 3 at pH 2.0 and 37 °C. The sequences of ODNs are listed in Table 1.

Figure 4

Inhibitory effect of Cho on depurination under various pH. (A) pH dependence of the inhibitory ratio I _r of Cho for the depurination of N30. (B) The rate constants of depurination in the presence and absence of Cho under various pH. N/P = 3 at 37 °C.

Figure 5

Inhibitory effect of Cho on the depurination of N30 in the presence of metal ions (orange bars). The blue bars are the inhibitory effect of metal ions only. N/P = 3 at pH 1.4 (HCl) and 37 °C.

Figure 6

Effect of Spm concentration on the rate of depurination from N30 at pH 3.0 and 37 °C. The inserted figure shows the inhibitory ratios. Structure of Spm is presented in the bottom panel.

Figure 7

Inhibitory effect of Spm on depurination under various pH. (A) pH dependence of the inhibitory ratio (I _r) of Spm for the depurination of N30 (N/P = 10). (B) The rate constants of depurination in the presence and absence of Spm under various pH. The I _r value at pH 2.5–4.8 was measured at 60 °C, whereas the value at pH 6.0–7.0 was at 37 °C, simply because of experimental convenience (note that the I _r value was kept almost unchanged between 35 °C and 60 °C). In (B), the pH profile of each of k _p and k ₀ was presented where all the values were measured at 60 °C.