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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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Examples of gel shift mobility assays of the dissociation of 2 and 3 from DNA sequence 5 at 3and 10 days. Each dissociation experiment was performed in triplicate.
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fig6: Examples of gel shift mobility assays of the dissociation of 2 and 3 from DNA sequence 5 at 3and 10 days. Each dissociation experiment was performed in triplicate.

Mentions: The dissociation rate constants for 2 and 3 were determined with DNA sequences 1–5 usinggel shift mobility assays as previously reported.34 Briefly, the tetraintercalator is incubated with a stoichiometricamount of 32P-labeled 24-mer containing a 14 bp bindingsite. DNA with a bound tetraintercalator displays a retarded gel mobilityso that bound and unbound radiolabeled DNA can be quantified usingnative polyacrylamide gel electrophoresis (PAGE) and autoradiography.For the dissociation rate constant analysis, complete initial bindingis confirmed by a gel shift, and then a 100-fold excess of unlabeledDNA 24-mer is added. The large excess of unlabeled DNA duplex willbind any dissociated tetraintercalator and thereby prevent reassociationwith radiolabeled DNA. The amount of bound versus unbound radiolabeledDNA is monitored over time (Figure 6), andthe data are fit to a monoexponential decay equation to derive dissociationrate constants (Table 1 and Figure S2 of the Supporting Information). Both 2 and 3 display dissociation rates that decrease in the followingorder: DNA sequence 3 > 4 > 2 > 1 > 5 (spanning a roughly30-fold rangeof values). Dissociation from a control sequence, a 24-mer DNA duplexwith no known tetraintercalator binding site (5′-CATTTAACAACATGTTGTTGGCTC-3′),was also analyzed. While the control sequence did display a gel shiftwhen incubated with a stoichiometric amount of 2 and 3, full association was never seen, and both were fully dissociatedfrom the control sequence within 1 h; therefore, reliable values couldnot be determined. For the purposes of comparison, the dissociationkinetics of compound 1 were also investigated with DNA sequence 5. The previously reported extraordinarily slowdissociation rate constant for binding of 1 to DNA sequence 1 is also shown in Table 1, revealing a >100-fold difference in dissociation rate constantsfor 1 with DNA sequences 1 and 5.


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

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

Examples of gel shift mobility assays of the dissociation of 2 and 3 from DNA sequence 5 at 3and 10 days. Each dissociation experiment was performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Examples of gel shift mobility assays of the dissociation of 2 and 3 from DNA sequence 5 at 3and 10 days. Each dissociation experiment was performed in triplicate.
Mentions: The dissociation rate constants for 2 and 3 were determined with DNA sequences 1–5 usinggel shift mobility assays as previously reported.34 Briefly, the tetraintercalator is incubated with a stoichiometricamount of 32P-labeled 24-mer containing a 14 bp bindingsite. DNA with a bound tetraintercalator displays a retarded gel mobilityso that bound and unbound radiolabeled DNA can be quantified usingnative polyacrylamide gel electrophoresis (PAGE) and autoradiography.For the dissociation rate constant analysis, complete initial bindingis confirmed by a gel shift, and then a 100-fold excess of unlabeledDNA 24-mer is added. The large excess of unlabeled DNA duplex willbind any dissociated tetraintercalator and thereby prevent reassociationwith radiolabeled DNA. The amount of bound versus unbound radiolabeledDNA is monitored over time (Figure 6), andthe data are fit to a monoexponential decay equation to derive dissociationrate constants (Table 1 and Figure S2 of the Supporting Information). Both 2 and 3 display dissociation rates that decrease in the followingorder: DNA sequence 3 > 4 > 2 > 1 > 5 (spanning a roughly30-fold rangeof values). Dissociation from a control sequence, a 24-mer DNA duplexwith no known tetraintercalator binding site (5′-CATTTAACAACATGTTGTTGGCTC-3′),was also analyzed. While the control sequence did display a gel shiftwhen incubated with a stoichiometric amount of 2 and 3, full association was never seen, and both were fully dissociatedfrom the control sequence within 1 h; therefore, reliable values couldnot be determined. For the purposes of comparison, the dissociationkinetics of compound 1 were also investigated with DNA sequence 5. The previously reported extraordinarily slowdissociation rate constant for binding of 1 to DNA sequence 1 is also shown in Table 1, revealing a >100-fold difference in dissociation rate constantsfor 1 with DNA sequences 1 and 5.

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