We show that groove recognition of nucleic acid triple helices can be achieved with aminosugars. Among these aminosugars, neomycin is the most effective aminoglycoside (groove binder) for stabilizing a DNA triple helix. It stabilizes both the T·A·T triplex and mixed-base DNA triplexes better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (T·A·T and mixed base) without any effect on the DNA duplex. The selectivity of neomycin likely originates from its potential and shape complementarity to the triplex Watson–Hoogsteen groove, making it the first molecule that selectively recognizes a triplex groove over a duplex groove. The groove recognition of aminoglycosides is not limited to DNA triplexes, but also extends to RNA and hybrid triple helical structures. Intercalator–neomycin conjugates are shown to simultaneously probe the base stacking and groove surface in the DNA triplex. Calorimetric and spectrosocopic studies allow the quantification of the effect of surface area of the intercalating moiety on binding to the triplex. These studies outline a novel approach to the recognition of DNA triplexes that incorporates the use of non-competing binding sites. These principles of dual recognition should be applicable to the design of ligands that can bind any given nucleic acid target with nanomolar affinities and with high selectivity.

Neomycin is the most effective aminoglycoside (groove binder) in stabilizing a DNA triple helix. It stabilizes TAT, as well as mixed base DNA triplexes, better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (in the presence of salt), without any effect on the DNA duplex. (1) Triplex stabilization by neomycin is salt dependent (increased KCl and MgCl(2) concentrations decrease neomycin's effectiveness, at a fixed drug concentration). (2) Triplex stabilization by neomycin is pH dependent (increased pH decreases neomycin's effectiveness, at a fixed drug concentration). (3) CD binding studies indicate approximately 5-7 base triplets/drug apparent binding site, depending upon the structure/sequence of the triplex. (4) Neomycin shows nonintercalative groove binding to the DNA triplex, as evident from viscometric studies. (5) Neomycin shows a preference for stabilization of TAT triplets but can also accommodate CGC(+) triplets. (6) Isothermal titration calorimetry (ITC) studies reveal an association constant of approximately 2 x 10(5) M(-)(1) between neomycin and an intramolecular triplex and a higher K(a) for polydA.2polydT. (7) Binding/modeling studies show a marked preference for neomycin binding to the larger W-H groove. Ring I/II amino groups and ring IV amines are proposed to be involved in the recognition process. (8) The novel selectivity of neomycin is suggested to be a function of its charge and shape complementarity to the triplex W-H groove, making neomycin the first molecule that selectively recognizes a triplex groove over a duplex groove.

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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 (Ultra-Violet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 towards 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 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 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’), × = hexaethylene glycol linker] result 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 towards DNA duplexes with alternate AT base pairs. (7) Salt dependent studies indicate the formation of 3 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 follow the 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 AT tract containing DNA duplex (B* DNA, d[5’-G3A5T5C3-3’]) with an 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 three orders of magnitude higher affinity than a GC rich DNA (A form, d[5’-A3G5C5T3-3’]).

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