The stabilization of poly(dA).2poly(dT) triplex, a 22-base DNA triplex, and poly(rA).2poly(rU) triple helix by neomycin is reported. The melting temperatures, the association and dissociation kinetic parameters, and activation energies (E(on) and E(off)) for the poly(dA).2poly(dT) triplex in the presence of aminoglycosides and other triplex binding ligands were determined by UV thermal analysis. Our results indicate that: (i) neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA).poly(dT) are virtually unaffected. (ii) Neomycin is the most active and triplex-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3-->2) vs DeltaT(m2)(-->)(1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3)(-->)(2) for a mixed 22-base DNA triplex containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of triplex (k(on)) are significantly enhanced upon increasing molar ratios of neomycin, making triplex association rates closer to duplex association rates. (v) E(on) values become more negative upon increasing concentration of aminoglycosides (paromomycin and neomycin). E(off) values do not show any change for most aminoglycosides except neomycin. (vi) Aminoglycosides can effectively stabilize RNA [poly(rA).2poly(rU)] triplex, with neomycin[being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.

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.

Aminoglycosides have been at the forefront of antimicrobial therapy for the past 50 years. Their specificity is believed to lie in binding duplex RNAs (rRNA). Competition dialysis studies of various nucleic acid forms with 9-aminoacridine, quinacrine, and a neomycin-acridine conjugate were carried out. Our results suggest a strong preference for aminoglycoside binding to nucleic acids that can adopt an A-type conformation. These results challenge the common belief that aminoglycoside specificity is simply for duplex RNAs.

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Recent developments have indicated that aminoglycoside binding is limited not to RNA but to nucleic acids that, like RNA, adopt conformations similar to the A-form. We have further sought to expand the utility of aminoglycoside binding to B-DNA structures by conjugating neomycin, an aminoglycoside antibiotic, with the B-DNA minor groove binding ligand Hoechst 33258. Described herein are novel neomycin-Hoechst 33258 conjugates developed for exploring B-DNA groove recognition. We have varied the two reported conjugates in linker length and composition in an effort to improve our understanding of the spatial differences that define B-DNA binding. Spectroscopic studies such as ultraviolet (UV) melting, isothermal fluorescence titrations, differential scanning calorimetry (DSC), and circular dichroism (CD) together illustrate the mode of binding by such conjugates. Both conjugates exhibit enhanced thermal stabilization of A.T rich duplexes when compared to Hoechst 33258.