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

(K34)₂Ni(II) shows different binding profiles towards wtTel26 and Tel26 at 25°C.

<p>ITC profiles corresponding to the titration of 25 µM wtTel26 (Panel A) or Tel26 (Panel B) with (K34)₂Ni(II) at 25°C in 10 mM Tris, 20 mM KCl, pH 7.5. Raw ITC data (top panel) and binding isotherms (bottom panel).</p

Thermodynamic parameters derived from ITC titrations describing the interaction of (K34)₂Ni(II) with Tel26 and wtTel26 at 25°C in 10 mM Tris, 20 mM KCl, pH 7.5.

*<p>: the applied model of sequential binding sites considers the binding events as exactly integral numbers. Reported data are the average of three independent measurements.</p

The energetic contribution for (K34)₂Ni(II) binding towards tested G-quadruplexes is differently modulated.

<p>Energetic contributions describing the interaction between tested G-quadruplexes and (K34)₂Ni(II) at 37°C in 10 mM Tris, 20 mM KCl, pH 7.5. The associated enthalpic variations (ΔH) are shown in black, the entropic ones (-TΔS) in white and the Gibbs energy changes (ΔG) in grey.</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

Effect of G-Quadruplex Polymorphism on the Recognition of Telomeric DNA by a Metal Complex

<div><p>The physiological role(s) played by G-quadruplexes renders these ‘non-canonical’ DNA secondary structures interesting new targets for therapeutic intervention. In particular, the search for ligands for selective recognition and stabilization of G-quadruplex arrangements has led to a number of novel targeted agents. An interesting approach is represented by the use of metal-complexes, their binding to DNA being modulated by ligand and metal ion nature, and by complex stoichiometry. In this work we characterized thermodynamically and stereochemically the interactions of a Ni(II) bis-phenanthroline derivative with telomeric G-quadruplex sequences using calorimetric, chiroptical and NMR techniques. We employed three strictly related sequences based on the human telomeric repeat, namely Tel22, Tel26 and wtTel26, which assume distinct conformations in potassium containing solutions. We were able to monitor specific enthalpy/entropy changes according to the structural features of the target telomeric sequence and to dissect the binding process into distinct events. Interestingly, temperature effects turned out to be prominent both in terms of binding stoichiometry and ΔH/ΔS contributions, while the final G-quadruplex-metal complex architecture tended to merge for the examined sequences. These results underline the critical choice of experimental conditions and DNA sequence for practical use of thermodynamic data in the rational development of effective G-quadruplex binders.</p> </div

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

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