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Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants.

Han YW, Matsumoto T, Yokota H, Kashiwazaki G, Morinaga H, Hashiya K, Bando T, Harada Y, Sugiyama H - Nucleic Acids Res. (2012)

Bottom Line: It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides.The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller.The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Honmachi, Sakyo, Kyoto 606-8501, Japan. han.yongwoon.4u@kyoto-u.ac.jp

ABSTRACT
N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py-Im polyamides 1-5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides. The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py-Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides. This fundamental knowledge of the binding of Py-Im polyamides to DNA will facilitate the design of hairpin Py-Im polyamides as synthetic DNA-binding modules.

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SPR sensorgrams for the interaction of Py–Im polyamides with hairpin DNAs immobilized on a sensor chip SA. (A) Py–Im polyamide 1 with ODN1 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (B) Py–Im polyamide 2 with ODN2 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (C) Py–Im polyamide 3 with ODN3 at a concentration range from 2.5 × 10−8 (lowest curve) to 4.0 × 10−7 M (highest curve). (D) Py–Im polyamide 4 with ODN4 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve). (E) Py–Im polyamide 5 with ODN5 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve).
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gks897-F3: SPR sensorgrams for the interaction of Py–Im polyamides with hairpin DNAs immobilized on a sensor chip SA. (A) Py–Im polyamide 1 with ODN1 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (B) Py–Im polyamide 2 with ODN2 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (C) Py–Im polyamide 3 with ODN3 at a concentration range from 2.5 × 10−8 (lowest curve) to 4.0 × 10−7 M (highest curve). (D) Py–Im polyamide 4 with ODN4 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve). (E) Py–Im polyamide 5 with ODN5 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve).

Mentions: To measure KD, ka and kd values of 1–5 for their target DNA, we performed SPR assays as described in the ‘Materials and Methods’ section. The five 5′-biotinylated hairpin DNAs (ODN1–5) (Figure 2) that include the target DNA sequences were immobilized on a streptavidin-coated sensor chip, and the Py–Im polyamide solutions were injected. As shown in Figure 3, SPR sensorgrams were obtained, and the kinetic binding parameters KD, ka and kd were determined (Table 1). The KDs of 1–5 increased with an increase in the number of Im as follows: 2.5 × 10−9, 3.7 × 10−9, 3.6 × 10−8, 4.8 × 10−8 and 5.4 × 10−8 M, respectively. Interestingly, the binding affinities of 1–4 were 22, 15, 1.5 and 1.1-fold, respectively, over that of 5. Of importance was that the kd values of 1–5 were comparable with each other (0.0039∼0.014 s−1) (Table 1), whereas the ka values of 1–5 were 1.5 × 106, 1.2 × 106, 2.8 × 105, 3.0 × 105 and 1.3 × 105 M−1s−1, respectively (Table 1). These results indicate that the association rate of the Py–Im polyamides with their target DNA decreased as the number of Im in the Py–Im polyamides increased. However, once 1–5 bound to their target DNAs, the dissociation rates of the Py–Im polyamides from the respective complexes were comparable with each other. We also determined the free energy change (ΔG°, kcal/M) from the KD on the formation of the Py–Im polyamides 1–5/DNA complexes (Table 1). The ΔG° values of 1/ODN1, 2/ODN2, 3/ODN3, 4/ODN4 and 5/ODN5 were −11.7, −11.5, −10.2, −10.0 and −9.9 kcal/M, respectively.Figure 3.


Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants.

Han YW, Matsumoto T, Yokota H, Kashiwazaki G, Morinaga H, Hashiya K, Bando T, Harada Y, Sugiyama H - Nucleic Acids Res. (2012)

SPR sensorgrams for the interaction of Py–Im polyamides with hairpin DNAs immobilized on a sensor chip SA. (A) Py–Im polyamide 1 with ODN1 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (B) Py–Im polyamide 2 with ODN2 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (C) Py–Im polyamide 3 with ODN3 at a concentration range from 2.5 × 10−8 (lowest curve) to 4.0 × 10−7 M (highest curve). (D) Py–Im polyamide 4 with ODN4 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve). (E) Py–Im polyamide 5 with ODN5 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve).
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Related In: Results  -  Collection

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Show All Figures
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gks897-F3: SPR sensorgrams for the interaction of Py–Im polyamides with hairpin DNAs immobilized on a sensor chip SA. (A) Py–Im polyamide 1 with ODN1 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (B) Py–Im polyamide 2 with ODN2 at a concentration range from 1.25 × 10−8 (lowest curve) to 2.0 × 10−7 M (highest curve). (C) Py–Im polyamide 3 with ODN3 at a concentration range from 2.5 × 10−8 (lowest curve) to 4.0 × 10−7 M (highest curve). (D) Py–Im polyamide 4 with ODN4 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve). (E) Py–Im polyamide 5 with ODN5 at a concentration range from 2.5 × 10−8 (lowest curve) to 8.0 × 10−7 M (highest curve).
Mentions: To measure KD, ka and kd values of 1–5 for their target DNA, we performed SPR assays as described in the ‘Materials and Methods’ section. The five 5′-biotinylated hairpin DNAs (ODN1–5) (Figure 2) that include the target DNA sequences were immobilized on a streptavidin-coated sensor chip, and the Py–Im polyamide solutions were injected. As shown in Figure 3, SPR sensorgrams were obtained, and the kinetic binding parameters KD, ka and kd were determined (Table 1). The KDs of 1–5 increased with an increase in the number of Im as follows: 2.5 × 10−9, 3.7 × 10−9, 3.6 × 10−8, 4.8 × 10−8 and 5.4 × 10−8 M, respectively. Interestingly, the binding affinities of 1–4 were 22, 15, 1.5 and 1.1-fold, respectively, over that of 5. Of importance was that the kd values of 1–5 were comparable with each other (0.0039∼0.014 s−1) (Table 1), whereas the ka values of 1–5 were 1.5 × 106, 1.2 × 106, 2.8 × 105, 3.0 × 105 and 1.3 × 105 M−1s−1, respectively (Table 1). These results indicate that the association rate of the Py–Im polyamides with their target DNA decreased as the number of Im in the Py–Im polyamides increased. However, once 1–5 bound to their target DNAs, the dissociation rates of the Py–Im polyamides from the respective complexes were comparable with each other. We also determined the free energy change (ΔG°, kcal/M) from the KD on the formation of the Py–Im polyamides 1–5/DNA complexes (Table 1). The ΔG° values of 1/ODN1, 2/ODN2, 3/ODN3, 4/ODN4 and 5/ODN5 were −11.7, −11.5, −10.2, −10.0 and −9.9 kcal/M, respectively.Figure 3.

Bottom Line: It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides.The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller.The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Honmachi, Sakyo, Kyoto 606-8501, Japan. han.yongwoon.4u@kyoto-u.ac.jp

ABSTRACT
N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py-Im polyamides 1-5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides. The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py-Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides. This fundamental knowledge of the binding of Py-Im polyamides to DNA will facilitate the design of hairpin Py-Im polyamides as synthetic DNA-binding modules.

Show MeSH
Related in: MedlinePlus