Subtle Recognition of 14-Base Pair DNA Sequences via Threading Polyintercalation

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 C2 symmetry in the DNA-binding molecule appeared to contribute to enhanced association rates.

Threading Polyintercalators with Extremely Slow Dissociation Rates and Extended DNA Binding Sites

“…This result matches DNase I footprinting of 1 with sequence A , which showed a footprint starting at 50 nM and full occupation at 100 nM. 21 At these concentrations, no non-specific binding was observed for 2 – 4 .…”

“…Dissociation results from this type of incubation match those of extended 37 °C incubations. 21 A recent study with a ruthenium monointercalator also suggested that the activation energy for intercalation is lowered with heat denatured DNA, resulting in faster intercalation processes. 26 Complete association between polyintercalator and duplex was confirmed by native PAGE, as all of the radioactively labeled duplex migrated at the expected slower rate.…”

“…9,21 These experiments exploit the retarded migration seen by polyacrylamide gel electrophoresis (PAGE) when polyintercalator is tightly bound to an oligonucleotide. For these experiments, a stoichiometric amount of intercalator and [ 32 P]-labeled DNA duplex were allowed to equilibrate by heating the DNA to 60 °C, adding intercalator, allowing the solution to cool slowly, and then holding at room temperature overnight.…”

“…Hexaintercalator 5 was synthesized in the same fashion as the tetraintercalators, and the Fmoc-(β-Ala) 3 -OH and Fmoc-(Gly) 3 -OH linkers were synthesized prior to SPPS in order to maximize yields. 21 …”

“…10,11 While threading mono- and bisintercalators continue to be of interest, 12–15 reports of longer polyintercalators, threading or not, are rare. 16–21 …”