Rt marker lane (M lanes). The symbol * indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL, with loss of CL. Position of alkylation is evinced by comparison of cleavage bands after piperidine treatment and the Maxam and Gilbert marker lane. Oligonucleotide sequences are indicated on the left of the 11089-65-9 site corresponding marker lane (M lanes). Base numbering has been assigned in the 5 primeR3 prime direction. doi:10.1371/journal.pone.0052994.gbulge length. Strikingly, the mobility of the 5- and 7-bulged oligonucleotides dramatically incremented and it remained only mildly lower than that of the control ss oligo (compare lanes 5 and 7 with ss, left side). In contrast, the number of non-paired bases in hairpin oligonucleotides only slightly influenced the oligonucleotide electrophoretic mobility, which barely decreased with increasing hairpin length (Fig. 6, right side).Discussion and ConclusionsNon-canonical nucleic acid structures have been postulated to mediate protein-nucleic acid interactions and frameshift mutations, some of which may result in a variety of diseases and cancers. Therefore, both recognition and elucidation of the conformation of unusual secondary structures would be of the utmost importance to predict unexpected biological effects generating at the genomic level. The natural compound CL had been previously shown to be able to detect and induce cleavage in partially ss regions within supercoiled plasmids, whereas it resulted completely inert versus ds nucleic acids [17,18]. Here we showed that 
CL could detect the presence of non-paired sequences, when at least one ss non-T base was available, in a number of non-canonical DNA conformations. In addition, the degree of CL reactivity towards DNA bases indicated the accessibility of ss-sites, therefore providing a 23977191 practical and simple tool to dissect unconventional DNA structures. In the case of bulges, site accessibility of ss nucleotides varied depending on the length of the bulge itself. By using CL, we were able to demonstrate that the highest base accessibility was obtained when 3-bulged bases were present, indicating that TGT or TCT bulges, with both 
A/T- or G/C-rich flanking ds sequences, protruded from the double-helix. Accessibility was reduced both in shorter (2 and 1 bases) and, unexpectedly, in longer 23727046 (5 and 7 bases) bulges. Longer bulges were shown to be less sterically hindered by EMSA; therefore they likely fold back on themselves or stack onto the double-helix, hindering access to reactive groups. These data complement previous analysis on bulge conformations. As a general rule, it was reported that in the case of one-base-bulges, the bulged purines stacked into the duplex [23,24,25,26], whereas bulged pyrimidines were either stacked in or CASIN site looped out into solution, depending upon the temperature and the flanking sequence [27,28,29,30]. In the case of three- and fivenucleotide DNA bulges, three and five unpaired A bases were found to be mostly stacked into the helix continuously with the flanking DNA and to induce a local kink in the DNA moleculeFigure 4. CL footprinting of bulged oligonucleotides. A) Oligonucleotides 1, 6 and 7 were heat denaturated and folded in the presence of the appropriate complementary sequences (1b rev, 1c rev, 1d rev, Table 1) to obtain the bulged G A/T rich oligonucleotides shown above the gel. B) Oligonucleotides 8, 9, 10, 11 and 12 were heat denaturated and folded in the presence of the appropriate complemen.Rt marker lane (M lanes). The symbol * indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL, with loss of CL. Position of alkylation is evinced by comparison of cleavage bands after piperidine treatment and the Maxam and Gilbert marker lane. Oligonucleotide sequences are indicated on the left of the corresponding marker lane (M lanes). Base numbering has been assigned in the 5 primeR3 prime direction. doi:10.1371/journal.pone.0052994.gbulge length. Strikingly, the mobility of the 5- and 7-bulged oligonucleotides dramatically incremented and it remained only mildly lower than that of the control ss oligo (compare lanes 5 and 7 with ss, left side). In contrast, the number of non-paired bases in hairpin oligonucleotides only slightly influenced the oligonucleotide electrophoretic mobility, which barely decreased with increasing hairpin length (Fig. 6, right side).Discussion and ConclusionsNon-canonical nucleic acid structures have been postulated to mediate protein-nucleic acid interactions and frameshift mutations, some of which may result in a variety of diseases and cancers. Therefore, both recognition and elucidation of the conformation of unusual secondary structures would be of the utmost importance to predict unexpected biological effects generating at the genomic level. The natural compound CL had been previously shown to be able to detect and induce cleavage in partially ss regions within supercoiled plasmids, whereas it resulted completely inert versus ds nucleic acids [17,18]. Here we showed that CL could detect the presence of non-paired sequences, when at least one ss non-T base was available, in a number of non-canonical DNA conformations. In addition, the degree of CL reactivity towards DNA bases indicated the accessibility of ss-sites, therefore providing a 23977191 practical and simple tool to dissect unconventional DNA structures. In the case of bulges, site accessibility of ss nucleotides varied depending on the length of the bulge itself. By using CL, we were able to demonstrate that the highest base accessibility was obtained when 3-bulged bases were present, indicating that TGT or TCT bulges, with both A/T- or G/C-rich flanking ds sequences, protruded from the double-helix. Accessibility was reduced both in shorter (2 and 1 bases) and, unexpectedly, in longer 23727046 (5 and 7 bases) bulges. Longer bulges were shown to be less sterically hindered by EMSA; therefore they likely fold back on themselves or stack onto the double-helix, hindering access to reactive groups. These data complement previous analysis on bulge conformations. As a general rule, it was reported that in the case of one-base-bulges, the bulged purines stacked into the duplex [23,24,25,26], whereas bulged pyrimidines were either stacked in or looped out into solution, depending upon the temperature and the flanking sequence [27,28,29,30]. In the case of three- and fivenucleotide DNA bulges, three and five unpaired A bases were found to be mostly stacked into the helix continuously with the flanking DNA and to induce a local kink in the DNA moleculeFigure 4. CL footprinting of bulged oligonucleotides. A) Oligonucleotides 1, 6 and 7 were heat denaturated and folded in the presence of the appropriate complementary sequences (1b rev, 1c rev, 1d rev, Table 1) to obtain the bulged G A/T rich oligonucleotides shown above the gel. B) Oligonucleotides 8, 9, 10, 11 and 12 were heat denaturated and folded in the presence of the appropriate complemen.