Dev P. Arya

Aminoglycoside Binding to Oxytricha nova Telomeric DNA

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ∼ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.

Molecular recognition of a DNA:RNA hybrid: sub-nanomolar binding by a neomycin-methidium conjugate.

A novel neomycin-methidium conjugate was synthesized. The covalent linkage of the aminoglycoside to an intercalator, a derivative of ethidium bromide, results in a new conjugate capable of selective recognition of the DNA:RNA hybrid duplex. Spectroscopic methods: UV, CD, fluorescence, and calorimetric techniques: DSC and ITC were used to characterize the sub-nanomolar binding displayed by the conjugate for the DNA:RNA hybrid duplex, poly(dA):poly(rU).

Triple recognition of B-DNA by a neomycin-Hoechst 33258-pyrene conjugate.

Recent developments have indicated that aminoglycoside binding is not limited to RNA, but to nucleic acids that, like RNA, adopt conformations similar to its A-form. We 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. Envisioning a dual groove binding mode, we have extended the potential recognition process to include a third, intercalative moiety. Similar conjugates, which vary in the number of binding moieties but maintain identical linkages to allow direct comparisons to be made, have also been prepared. We report herein novel neomycin- and Hoechst 33258-based conjugates developed in our laboratories for exploring the recognition potential with B-DNA. Spectroscopic studies such as UV melting, differential scanning calorimetry, isothermal fluorescence titrations, and circular dichroism together illustrate the triple recognition of the novel conjugate containing neomycin, Hoechst 33258, and pyrene. This study represents the first example of DNA molecular recognition capable of minor versus major groove recognition in conjunction with intercalation.

DNA Triple Helix Stabilization by Aminoglycoside Antibiotics

The stabilization of the poly(dA) x 2poly(dT) triple helix by neomycin is reported. Preliminary results indicate that neomycin stabilizes DNA triple helices and the double helical structures composed of poly(dA) x poly(dT) are virtually unaffected. This is the first report of the interaction of aminoglycoside antibiotics with DNA triple helices.

An Expanding View of Aminoglycoside–Nucleic Acid Recognition

Publisher Summary This chapter discusses the applications of aminoglycosides in the field of medicine. The origin of aminoglycoside antibiotics began with streptomycin 60 years ago. Isolated from Actinomyces griseus, streptomycin immediately found applications for the treatment against the tuberculosis-causing microorganism—Mycobacterium tuberculosis. This was the second antibiotic after penicillin to be used clinically. Throughout the coming years, a number of aminoglycosides were discovered with varying potencies for treating infections. However, therapeutic applications were diminished by the emergence of resistance to aminoglycosides by bacteria. Though widely known for binding to prokaryotic ribosomal RNA, aminoglycosides have more recently shown affinity for other nucleic acid targets. This opens door to a new area of therapeutic applications. This chapter outlines the history of aminoglycosides, from their discovery, structural elucidation, and mechanism of action to the current knowledge of mechanisms of action and resistance, toxicity, and novel nucleic acid targets. Owing to a number of recent reviews pertaining to some of these areas, particular emphasis is placed on the recently discovered aminoglycoside targets and their potential therapeutic applications.

Molecular recognition of single-stranded RNA: Neomycin binding to poly(A)

Poly(A) is a relevant sequence in cell biology due to its importance in mRNA stability and translation initiation. Neomycin is an aminoglycoside antibiotic that is well known for its ability to target various nucleic acid structures. Here it is reported that neomycin is capable of binding tightly to a single‐stranded oligonucleotide (A30) with a Kd in the micromolar range. CD melting experiments support complex formation and indicate a melting temperature of 47 °C. The poly(A) duplex, which melts at 44 °C (pH 5.5), was observed to melt at 61 °C in the presence of neomycin, suggesting a strong stabilization of the duplex by the neomycin.

Synthesis of aminoglycoside-DNA conjugates.

Development of aminoglycoside-nucleic acid conjugates is presented. Synthesis of a DNA dimer covalently linked to kanamycin and neomycin isothiocyanates has been carried out. The development of such conjugates will help couple the sequence specificity of nucleic acids to the electrostatic/shape complementarity of aminoglycoside antibiotics in binding nucleic acid targets.

Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.

Targeting C-myc G-Quadruplex: Dual Recognition by Aminosugar-Bisbenzimidazoles with Varying Linker Lengths

G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.

Triple recognition of B-DNA.

A novel conjugate of Hoechst 33258, pyrene and neomycin was synthesized and examined for its binding and stabilization of A-T rich DNA duplexes using spectroscopic and viscometric techniques. The conjugate, containing three well known ligands that bind nucleic acids albeit in different binding modes, was found to significantly stabilize DNA over parent conjugates containing only one or both of the other recognition elements. The study represents the first example of DNA molecular recognition capable of minor/major groove recognition in conjunction with intercalation.