RNA−Ligand Interactions: Affinity and Specificity of Aminoglycoside Dimers and Acridine Conjugates to the HIV-1 Rev Response Element

Abstract

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified “monomeric” aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K_d ≤ 10 nM), but few compounds have a high specificity for the RRE. Compared to the acridine conjugates, the dimeric and unmodified aminoglycosides exhibit good RNA over DNA selectivity, but show little differentiation between different RNA molecules. Neomycin-based derivatives consistently have the highest RNA and DNA affinities, but the lowest RRE specificity. To optimize these derivatives for RRE specificity, a series of neomycin−acridine conjugates with variable linker lengths were synthesized and evaluated. The neo-acridine conjugate with the shortest linker length has the optimal RRE specificity. Duplex DNA, on the other hand, prefers the acridine conjugate with the longest linker length, and duplex RNA (poly r[A]−r[U]) has the highest affinity for the conjugate with an intermediate linker length. Compared to neomycin B, the derivatives based upon tobramycin and kanamycin A have slightly lower RRE affinities, but better RRE specificities. These results illustrate how the binding affinity and specificity of aminoglycoside intercalator conjugates can be tuned by optimizing the linker length and by changing the identity of the aminoglycoside moiety. These results also indicate that many aminoglycoside-based ligands are capable of high-affinity binding of RNA, but achieving high site specificity remains a challenging objective.