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Neomycin−Neomycin Dimer: An All-Carbohydrate Scaffold with High Affinity for AT-Rich DNA Duplexes

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journal contribution
posted on 18.05.2011, 00:00 by Sunil Kumar, Liang Xue, Dev P. Arya
A dimeric neomycin−neomycin conjugate 3 with a flexible linker, 2,2′-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10−12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5′-A12-x-T12-3′), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ∼108 M−1 at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5′-A12-x-T12-3′] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5′-G3A5T5C3-3′] > AT alternate, d[5′-G3(AT)5C3-3′] > GC-rich d[5′-A3G5C5T3-3′]. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5′-G3A5T5C3-3′]) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5′-G3(AT)5C3-3′]) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5′-A3G5C5T3-3′]).