Molecular Determinants for DNA Minor Groove Recognition:  Design of a Bis-Guanidinium Derivative of Ethidium That Is Highly Selective for AT-Rich DNA Sequences

Abstract

The phenanthridinium dye ethidium bromide is a prototypical DNA intercalating agent. For decades, this anti-trypanosomal agent has been known to intercalate into nucleic acids, with little preference for particular sequences. Only polydA-polydT tracts are relatively refractory to ethidium intercalation. In an effort to tune the sequence selectivity of known DNA binding agents, we report here the synthesis and detailed characterization of the mode of binding to DNA of a novel ethidium derivative possessing two guanidinium groups at positions 3 and 8. This compound, DB950, binds to DNA much more tightly than ethidium and exhibits distinct DNA-dependent absorption and fluorescence properties. The study of the mode of binding to DNA by means of circular and electric linear dichroism revealed that, unlike ethidium, DB950 forms minor groove complexes with AT sequences. Accurate quantification of binding affinities by surface plasmon resonance using A(n)T(n) hairpin oligomer indicated that the interaction of DB950 is over 10-50 times stronger than that of ethidium and comparable to that of the known minor groove binder furamidine. DB950 interacts weakly with GC sites by intercalation. DNase I footprinting experiments performed with different DNA fragments established that DB950 presents a pronounced selectivity for AT-rich sites, identical with that of furamidine. The replacement of the amino groups of ethidium with guanidinium groups has resulted in a marked gain of both affinity and sequence selectivity. DB950 provides protection against DNase I cleavage at AT-containing sites which frequently correspond to regions of enhanced cleavage in the presence of ethidium. Although DB950 maintains a planar phenanthridinium chromophore, the compound no longer intercalates at AT sites. The guanidinium groups of DB950, just like the amidinium group of furamidine (DB75), are the critical determinants for recognition of AT binding sites in DNA. The chemical modulation of the ethidium exocyclic amines is a profitable option to tune the nucleic acid recognition properties of phenylphenanthridinium dyes.

Survey of the year 2005 commercial optical biosensor literature

“…These articles exemplify today's state-of-the-art applications in biosensor studies and range from wellexecuted, qualitative [184,294], kinetic [429, 475,664,704,776,779,906,907], and equilibrium analyses [664,751,906,907] to the use of novel assay designs [526] and new technology [583]. Even more importantly, these articles illustrate the quality of data and interpretation that we expect to see in biosensor-based studies.…”

The phenanthridine biguanides efficiently differentiate between dGdC, dAdT and rArU sequences by two independent, sensitive spectroscopic methods

“…[9][10][11][12] Also, recent study demonstrated that double amine-guanidine substitution at the 3 and 8 positions of the ethidium bromide changed the original intercalator into a minor groove binder of DNA. 13 Moreover, a single amineguanidine exchange in known intercalators (anthracene, pyrene, tiophene, fluorene) resulted in a marked gain of affinity and antiproliferative activity while keeping their intercalative mode of binding.…”

Novel bis-phenanthridine derivatives with easily tunable linkers, study of their interactions with DNA and screening of antiproliferative activity

“…[1][2][3] In addition, it was shown that the chemical modulation of the ethidium substituents is a profitable option to tune the nucleic acid recognition properties of phenanthridine dyes. [4] Very recent reports about numerous applications point toward versatility of the phenanthridine core, [5][6][7][8][9] including even intriguing biological activity. [10] A huge number of bis-phenanthridine derivatives were prepared with the aim of not only enhanced affinity due to the bis-intercalation into DNA/RNA but also with the idea of introducing selectivity.…”