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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 1–3 with DNA sequence1 and of 1 with DNA sequence 5. Lane1 contained no DNase I. Lane 2 containedan adenine-specific cleavage reaction mixture.37 Lane 3 contained no intercalator. Tetraintercalator concentrationsfor lanes 4–7 were 50, 100, 150, and 200 nM, respectively.
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fig5: Concentration-dependentDNase I footprints of 1–3 with DNA sequence1 and of 1 with DNA sequence 5. Lane1 contained no DNase I. Lane 2 containedan adenine-specific cleavage reaction mixture.37 Lane 3 contained no intercalator. Tetraintercalator concentrationsfor lanes 4–7 were 50, 100, 150, and 200 nM, respectively.

Mentions: DNase I footprinting studies were also performedwith 2 and 3 on DNA containing the preferredpalindromic bindingsite for 1, DNA sequence 1, and with 1 on DNA containing the most promising target for 2 and 3, DNA sequence 5 (Figure 5). The strong footprint of 1 with DNA sequence 1 is also shown for reference. Perhaps surprisingly,for DNA sequence 1, both 2 and 3 display significant footprints. Compound 2 begins tooccupy the site at 100 nM, and 3 begins to bind at 50nM, ranking these interactions the second strongest seen in the study,behind only those seen with DNA sequence 5. For tetraintercalator 1, some binding was seen with DNA sequence 5,with a footprint beginning to appear at 100 nM, although the siteis never fully occupied in the concentration range tested. For 2 and 3 at higher concentrations, other siteson the DNA, generally GC-rich regions, also seem to be occupied, implyingthat 2 and 3 may be less specific in bindingcompared to 1 in the context of these sequences. Thisis perhaps not altogether too surprising given that NDI monointercalatorsprefer binding GC-rich DNA.28


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 1–3 with DNA sequence1 and of 1 with DNA sequence 5. Lane1 contained no DNase I. Lane 2 containedan adenine-specific cleavage reaction mixture.37 Lane 3 contained no intercalator. Tetraintercalator concentrationsfor 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

fig5: Concentration-dependentDNase I footprints of 1–3 with DNA sequence1 and of 1 with DNA sequence 5. Lane1 contained no DNase I. Lane 2 containedan adenine-specific cleavage reaction mixture.37 Lane 3 contained no intercalator. Tetraintercalator concentrationsfor lanes 4–7 were 50, 100, 150, and 200 nM, respectively.
Mentions: DNase I footprinting studies were also performedwith 2 and 3 on DNA containing the preferredpalindromic bindingsite for 1, DNA sequence 1, and with 1 on DNA containing the most promising target for 2 and 3, DNA sequence 5 (Figure 5). The strong footprint of 1 with DNA sequence 1 is also shown for reference. Perhaps surprisingly,for DNA sequence 1, both 2 and 3 display significant footprints. Compound 2 begins tooccupy the site at 100 nM, and 3 begins to bind at 50nM, ranking these interactions the second strongest seen in the study,behind only those seen with DNA sequence 5. For tetraintercalator 1, some binding was seen with DNA sequence 5,with a footprint beginning to appear at 100 nM, although the siteis never fully occupied in the concentration range tested. For 2 and 3 at higher concentrations, other siteson the DNA, generally GC-rich regions, also seem to be occupied, implyingthat 2 and 3 may be less specific in bindingcompared to 1 in the context of these sequences. Thisis perhaps not altogether too surprising given that NDI monointercalatorsprefer binding GC-rich DNA.28

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