CL footprinting of nicked oligonucleotides.

<p>A) Oligonucleotides 17, 25 and 26 were heat denaturated and folded in the presence of the appropriate complementary sequences (15 rev-16 rev, 17 rev-18 rev, 19 rev-18 rev, respectively, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>). B) Oligonucleotide 24 was heat denaturated and folded in the presence of the appropriate complementary sequences (12 rev-12 rev, 11 rev-13 rev, 14 rev-13 rev, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>) to obtain the nicked C A/T rich oligonucleotides shown above the gel. The folded oligonucleotides were incubated with CL (100 µM) for 24 h at 37°C. After reaction, samples were precipitated and either kept on ice or treated with hot piperidine and lyophilized (samples indicated by the symbol P) and loaded on a 20% denaturing polyacrylamide gel. The symbol § indicates CL/full-length DNA adducts which migrate slower than the full-length DNA. The symbol ¤ indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL. The symbol * indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL, with loss of CL. The symbols ÷ and # indicate bases in the ds region flanking the nicked moiety that are 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 aside of the corresponding marker lane (M lanes). Base numbering has been assigned in the 5 prime→3 prime direction.</p

CL footprinting of hairpin oligonucleotides.

<p>A) Oligonucleotides 37, 38, 39 and 40, B) oligonucleotides 41, 42, 43 and 44 and C) oligonucleotides 27, 30, 31, 29 and 28 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>) were heat denaturated and folded to obtain the hairpin G, C or T oligonucleotides shown above the gels. The folded oligonucleotides were incubated with CL (100 µM) for 24 h at 37°C. After reaction, samples were precipitated and either kept on ice or treated with hot piperidine and lyophilized (samples indicated by the symbol P) and loaded on a 20% denaturing polyacrylamide gel. The symbol * indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL, without or with loss of CL, at the G or C base exposed in the hairpin region. The symbol ¤ indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL, without or with loss of CL, at bases in the ds stem region of the oligonucleotide. 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 prime→3 prime direction.</p

Reagents used in this study.

<p>A) Chemical structure of CL. B) Schematic representation of the single-stranded (ss) regions of the oligonucleotides used, subdivided according to the secondary structure category. Double-stranded regions flanking the ss moiety are shown, because CL reactivity was assayed and compared towards oligonucleotides with both G/C and A/T-rich flanking regions. Arrows indicate the position of CL alkylation and cleavage. The size of the arrows corresponds to the degree of reactivity.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

CL footprinting of mismatched oligonucleotides.

<p>Oligonucleotides 5, and 1 were heat denaturated and folded in the presence of the appropriate complementary sequences (1a rev, 2 rev, 3 rev, respectively, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>) to obtain MM C/A, MM TG/TC and MM TGT/GTC oligonucleotides. The folded oligonucleotides were incubated with increasing concentrations (50–100 µM) of CL for 24 h at 37°C. After reaction, samples were precipitated and either kept on ice or treated with hot piperidine and lyophilized (samples indicated by the symbol P) and loaded on a 20% denaturing polyacrylamide gel. The symbol § indicates CL/full-length DNA adducts which migrate slower than the full-length DNA. The symbol ¤ indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL. CL is still bound to the cleaved oligonucleotide, thus the cleavage band runs slower than the corresponding band in the Maxam and Gilbert 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 prime→3 prime direction.</p

CL footprinting of bulged oligonucleotides.

<p>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, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>) 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 complementary sequences (8 rev, 9 rev, 10 rev, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>) to the bulged G G/C rich oligonucleotides shown above the gel. C) Oligonucleotides 5, 1, 13, and 14, were heat denaturated and folded in the presence of the appropriate complementary sequences (1b rev, 1c rev, 1d rev, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>) to obtain the bulged C A/T rich oligonucleotides shown above the gel. The folded oligonucleotides were incubated with increasing concentrations (50–100 µM) of CL for 24 h at 37°C. After reaction, samples were precipitated and either kept on ice or treated with hot piperidine and lyophilized (samples indicated by the symbol P) and loaded on a 20% denaturing polyacrylamide gel. The symbol § indicates CL/full-length DNA adducts which migrate slower than the full-length DNA. The symbol ¤ indicates bands that correspond to the oligonucleotide alkylated and cleaved by CL. CL is still bound to the cleaved oligonucleotide, thus the cleavage band runs slower than the corresponding band in the Maxam and Gilbert 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 prime→3 prime direction.</p

EMSA analysis of bulge and hairpin oligonucleotides.

<p>Oligonucleotides 50, 49, 48, 47, 46 and 45 were annealed to the appropriate complementary oligonucleotides (50 rev, 49 rev, 48 rev, 47 rev, 46 rev and 45 rev, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052994#pone-0052994-t001" target="_blank">Table 1</a>) to form ds, 1-, 2-, 3-, 5-, and 7-base bulged sequences, respectively. Oligonucleotides 54, 53, 52 and 51 were folded to form 3-, 5-, 7-, 9-base hairpin sequences. Ds and ss oligonucleotides with the same length as bulge or hairpin sequences were employed as controls of migration and they are indicated by the arrows aside the gel images.</p

Frequency of the occurrence of the hydrogen bonds.

<p>Frequency of the occurrence of the hydrogen bonds (HBs), monitored over 2 ns, every 10 ps, in the guanine core in the presence of C3 and A1T3 mutated G-quadruplex sequences. </p

RMSd conformation of C3 and A1T3 mutated G-quadruplex sequences.

<p>Lowest (A) and highest (B) RMSd conformation of C3 and A1T3 mutated G-quadruplex sequences during 2 ns MD simulations with respect to the starting structure (2HY9). The DNA is shown in purple (C3) and aquamarine (A1T3) wireframe rendering and its strand as orange cartoon. The K⁺ coordinating ions are represented as blue spheres. The RMSd value of each conformation is reported and expressed in Å. </p

Thermal difference spectra (TDS) and TDS factor plots of three representative oligonucleotides.

<p>A) TDS of C1, C2 and C3 sequences; B) TDS factors of C1, C2 and C3 sequences. TDS and TDS factors of all oligonucleotides are available in Figure S8 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084113#pone.0084113.s004" target="_blank">File S4</a>.</p