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Subtle recognition of 14-base pair DNA sequences via threading polyintercalation.

Smith AR, Ikkanda BA, Holman GG, Iverson BL - Biochemistry (2012)

Bottom Line: Chem. 3, 875-881].Herein are described new NDI-based tetraintercalators with a different major groove-binding module and a reversed N to C directionality of one of the minor groove-binding modules.DNase I footprinting and kinetic analyses revealed that these new tetraintercalators are able to discriminate, by as much as 30-fold, 14 bp DNA binding sites that differ by 1 or 2 bp.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, United States.

ABSTRACT
Small molecules that bind DNA in a sequence-specific manner could act as antibiotic, antiviral, or anticancer agents because of their potential ability to manipulate gene expression. Our laboratory has developed threading polyintercalators based on 1,4,5,8-naphthalene diimide (NDI) units connected in a head-to-tail fashion by flexible peptide linkers. Previously, a threading tetraintercalator composed of alternating minor-major-minor groove-binding modules was shown to bind specifically to a 14 bp DNA sequence with a dissociation half-life of 16 days [Holman, G. G., et al. (2011) Nat. Chem. 3, 875-881]. Herein are described new NDI-based tetraintercalators with a different major groove-binding module and a reversed N to C directionality of one of the minor groove-binding modules. DNase I footprinting and kinetic analyses revealed that these new tetraintercalators are able to discriminate, by as much as 30-fold, 14 bp DNA binding sites that differ by 1 or 2 bp. Relative affinities were found to correlate strongly with dissociation rates, while overall C(2) symmetry in the DNA-binding molecule appeared to contribute to enhanced association rates.

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Concentration-dependentDNase I footprints of 2 and 3 with DNA sequences 2–5. Lane 1 containedno DNase I. Lane 2 contained an adenine-specific cleavage reactionmixture.37 Lane 3 contained no intercalator.Tetraintercalator concentrations for lanes 4–7 were 50, 100,150, and 200 nM, respectively.
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fig4: Concentration-dependentDNase I footprints of 2 and 3 with DNA sequences 2–5. Lane 1 containedno DNase I. Lane 2 contained an adenine-specific cleavage reactionmixture.37 Lane 3 contained no intercalator.Tetraintercalator concentrations for lanes 4–7 were 50, 100,150, and 200 nM, respectively.

Mentions: Concentration-dependent DNaseI footprinting was used to evaluate binding of 2 and 3 to DNA sequences 2–5 (Figure 4). In these studies, the DNA was heated to 80 °C,and then the intercalator was added followed by slow cooling. Notethat footprints obtained in this way were identical to those of a4 day incubation at 37 °C, providing confidence that equilibriumhad been reached. To compare rigorously the binding to each sequence,the DNA used for footprinting contained only one of the proposed bindingsites with the same flanking sequences on either side (Supporting Information). Intercalators 2 and 3 bind DNA sequence 3 withthe lowest affinity and appear to never occupy the entire site. For DNA sequence 4, 2 begins to bind at 150 nM butnever fully occupies the sequence in the concentration range tested,and 3 begins to occupy the site at 100 nM. For DNA sequence 2, 2 begins to occupy the site at100 nM, while 3 gives attenuation of digestion bandsin the binding site starting at 50 nM. For DNA sequence 5, the binding site is mostly bound for both 2 and 3 at 50 nM. Thus, while all the target DNA sequences seemto experience some binding, the footprints seen with DNA sequence5 are the most distinct and occur at the lowest concentrationsfor both 2 and 3.


Subtle recognition of 14-base pair DNA sequences via threading polyintercalation.

Smith AR, Ikkanda BA, Holman GG, Iverson BL - Biochemistry (2012)

Concentration-dependentDNase I footprints of 2 and 3 with DNA sequences 2–5. Lane 1 containedno DNase I. Lane 2 contained an adenine-specific cleavage reactionmixture.37 Lane 3 contained no intercalator.Tetraintercalator concentrations for lanes 4–7 were 50, 100,150, and 200 nM, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369501&req=5

fig4: Concentration-dependentDNase I footprints of 2 and 3 with DNA sequences 2–5. Lane 1 containedno DNase I. Lane 2 contained an adenine-specific cleavage reactionmixture.37 Lane 3 contained no intercalator.Tetraintercalator concentrations for lanes 4–7 were 50, 100,150, and 200 nM, respectively.
Mentions: Concentration-dependent DNaseI footprinting was used to evaluate binding of 2 and 3 to DNA sequences 2–5 (Figure 4). In these studies, the DNA was heated to 80 °C,and then the intercalator was added followed by slow cooling. Notethat footprints obtained in this way were identical to those of a4 day incubation at 37 °C, providing confidence that equilibriumhad been reached. To compare rigorously the binding to each sequence,the DNA used for footprinting contained only one of the proposed bindingsites with the same flanking sequences on either side (Supporting Information). Intercalators 2 and 3 bind DNA sequence 3 withthe lowest affinity and appear to never occupy the entire site. For DNA sequence 4, 2 begins to bind at 150 nM butnever fully occupies the sequence in the concentration range tested,and 3 begins to occupy the site at 100 nM. For DNA sequence 2, 2 begins to occupy the site at100 nM, while 3 gives attenuation of digestion bandsin the binding site starting at 50 nM. For DNA sequence 5, the binding site is mostly bound for both 2 and 3 at 50 nM. Thus, while all the target DNA sequences seemto experience some binding, the footprints seen with DNA sequence5 are the most distinct and occur at the lowest concentrationsfor both 2 and 3.

Bottom Line: Chem. 3, 875-881].Herein are described new NDI-based tetraintercalators with a different major groove-binding module and a reversed N to C directionality of one of the minor groove-binding modules.DNase I footprinting and kinetic analyses revealed that these new tetraintercalators are able to discriminate, by as much as 30-fold, 14 bp DNA binding sites that differ by 1 or 2 bp.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712, United States.

ABSTRACT
Small molecules that bind DNA in a sequence-specific manner could act as antibiotic, antiviral, or anticancer agents because of their potential ability to manipulate gene expression. Our laboratory has developed threading polyintercalators based on 1,4,5,8-naphthalene diimide (NDI) units connected in a head-to-tail fashion by flexible peptide linkers. Previously, a threading tetraintercalator composed of alternating minor-major-minor groove-binding modules was shown to bind specifically to a 14 bp DNA sequence with a dissociation half-life of 16 days [Holman, G. G., et al. (2011) Nat. Chem. 3, 875-881]. Herein are described new NDI-based tetraintercalators with a different major groove-binding module and a reversed N to C directionality of one of the minor groove-binding modules. DNase I footprinting and kinetic analyses revealed that these new tetraintercalators are able to discriminate, by as much as 30-fold, 14 bp DNA binding sites that differ by 1 or 2 bp. Relative affinities were found to correlate strongly with dissociation rates, while overall C(2) symmetry in the DNA-binding molecule appeared to contribute to enhanced association rates.

Show MeSH
Related in: MedlinePlus