br Materials and methods br Results

Materials and methods
Results
Discussion A significant decrease in fluorescence was observed after the treatment of pure DNA with alkylating compounds. This decrease was characterized by a dose-response relationship. Mass spectrometry and additional fluorescence measurements ruled out the covalent modification of EthBr by SM. The alkylation of DNA by SM results in around 14–20% of DNA crosslinks [3], [19], [20]; up to 50% of these crosslinks are assumed to represent interstrand crosslinks [21]. We thus assumed that DNA crosslinks may have caused DNA condensation and impaired the access of the fluorescent dyes to the DNA. Further experiments using bifunctional N-alkylating agents (i.e. HN-3) revealed an even more pronounced decrease in fluorescence, which underlined our hypothesis. However, some bifunctional and crosslinking agents (i.e. HN-1, HN-2) had only a minor effect on fluorescence. These agents were shown to exhibit slower kinetics with regard to DNA alkylation [14], [22], [23], [24]. This may also be a reason for the lower toxicity of these compounds [25]. We thus assumed that, with regard to HN-1 and HN-2, lower reactivity was the reason for the only minor effects that we observed in our experiments. In order to counteract DNA condensation, we used restriction enzymes [26] to cleave alkylated DNA into small fragments. DNA fragments were thought to be more easily accessible for DNA dyes. Our results indeed demonstrated that the use of Biperiden HCl receptor enzymes restored the fluorescence signal of stained, alkylated DNA. The decrease in fluorescence intensity may, however, also be due to quenching effects. It is reported that especially chloride ions (Cl−) may impact fluorescence [27]. A single SM molecule is able to release two Cl− ions either due to SM hydrolysis or during DNA alkylation [2], [28]. In addition to the release of Cl− from the SM molecule, two protons per SM molecule are also produced, which results in the formation of hydrochloric acid [2], [29], [30], [31]. To mimic this fact, we performed additional experiments using NaCl or HCl instead of alkylating compounds. While NaCl had no effect on fluorescence, the use of HCl decreased fluorescence in a manner comparable to the alkylating compounds. Based on the results, we concluded that neither crosslinks nor Cl− but instead H+ were responsible for decreasing the fluorescence of SM-exposed DNA in aqueous, unbuffered solutions. This hypothesis was confirmed by repeating the experiments and using defined buffer systems (e.g. PBS, Tris-HCl) instead of water. In this case, the decrease in fluorescence was clearly prevented, especially when we used PBS or Tris-HCl. Other buffer systems that we tested were also suitable but were less effective.
Introduction In addition to the canonical double stranded structure, DNA can form various higher order structures such as bulges, and various kinds of mismatches, triplexes, to the G-quadruplex (G4). Over the past decades, accumulating evidence has begun to emerge that these non-canonical structures have important functional roles in genetic regulation and are correlated to many diseases.2, 3 For instance, the T-T mismatch formation from expandable repeat CTG sequences is implicated in human neurological diseases such as the myotonic dystrophy type 1 (DM1).