Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common platform fragment-based drug discovery, two fragments that bind to adjacent sites on a target are identified and are then linked together using different linkers to identify the linkage for optimum activity. What are not known from these studies are the effects these linkers, which typically contain C, H, and O atoms, have on the properties of the individual fragment. Herein, we investigate such effects in a bisbenzimidazole fragment whose derivatives have a wide range of therapeutic applications in nucleic acid recognition, sensing, and photodynamic therapy and as cellular probes. We report a dramatic effect of linker length and composition of alkynyl (clickable) Hoechst 33258 derivatives in target binding and cell uptake. We show that the binding of Hoechst 33258-modeled bisbenzimidazoles (1–9) that contain linkers of varying lengths (3–21 atoms) display length- and composition-dependent variation in B-DNA stabilization using a variety of spectroscopic methods. For a dodecamer DNA duplex, the thermal stabilization varied from 0.3 to 9.0 °C as the linker length increased from 3 to 21 atoms, respectively. Compounds with linker lengths of ≤11 atoms (such as compounds 1 and 5) are localized in the nucleus, while compounds with long linkers (such as compounds 8 and 9) are distributed in the extranuclear space, as well, with possible interactions with extranuclear targets. These findings provide insights into future drug design by revealing how linkers can influence the biophysical and cellular properties of individual drug fragments.

Recognition of nucleic acids remains an important endeavor in biology. Nucleic acids adopt shapes ranging from A-form (RNA and GC rich DNA) to B-form (AT rich DNA). We show, in this contribution, shape-specific recognition of A–U rich RNA duplex by a neomycin (Neo)–polydiacetylene (PDA) complex. PDA assemblies are fabricated by using a well-known diacetylene (DA) monomer, 10,12-pentacosadiynoic acid (PCDA). The response of poly(PCDA) assemblies is generated by mixing with a modified neomycin–PCDA monomer (Neo–PCDA). The functionalization by neomycin moiety provides specific binding with homopolyribonucleotide poly(rA)–poly(rU) stimulus. Various types of alcohols are utilized as additives to enhance the sensitivity of poly(PCDA)/Neo–PCDA assemblies. A change of absorption spectra is clearly observed when a relatively low concentration of poly(rA)–poly(rU) is added into the system. Furthermore, poly(PCDA)/Neo–PCDA shows a clear specificity for poly(rA)–poly(rU) over the corresponding DNA duplex. The variation of linker between neomycin moiety and conjugated PDA backbone is found to significantly affect its sensitivity. We also investigate other parameters including the concentration of Neo–PCDA and the DA monomer structure. Our results provide here preliminary data for an alternative approach to improve the sensitivity of PDA utilized in biosensing and diagnostic applications.

The antibacterial effects of aminoglycosides are based on their association with the A-site of bacterial rRNA and interference with the translational process in the bacterial cell, causing cell death. The clinical use of aminoglycosides is complicated by resistance and side effects, some of which arise from their interactions with the human mitochondrial 12S rRNA and its deafness-associated mutations, C1494U and A1555G. We report a rapid assay that allows screening of aminoglycoside compounds to these classes of rRNAs. These screening tools are important to find antibiotics that selectively bind to the bacterial A-site rather than human, mitochondrial A-sites and its mutant homologues. Herein, we report our preliminary work on the optimization of this screen using 12 anthraquinone–neomycin (AMA−NEO) conjugates against molecular constructs representing five A-site homologues, Escherichia coli, human cytosolic, mitochondrial, C1494U, and A1555G, using a fluorescent displacement screening assay. These conjugates were also tested for inhibition of protein synthesis, antibacterial activity against 14 clinically relevant bacterial strains, and the effect on enzymes that inactivate aminoglycosides. The AMA–NEO conjugates demonstrated significantly improved resistance against aminoglycoside-modifying enzymes (AMEs), as compared with NEO. Several compounds exhibited significantly greater inhibition of prokaryotic protein synthesis as compared to NEO and were extremely poor inhibitors of eukaryotic translation. There was significant variation in antibacterial activity and MIC of selected compounds between bacterial strains, with Escherichia coli, Enteroccocus faecalis, Citrobacter freundii, Shigella flexneri, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae, Staphylococcus epidermidis, and Listeria monocytogenes exhibiting moderate to high sensitivity (50–100% growth inhibition) whereas Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiellla pneumoniae, and MRSA strains expressed low sensitivity, as compared to the parent aminoglycoside NEO.Keywords: aminoglycoside modifying enzymes; antibacterial activity; translation inhibition;

Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs. A novel neomycin dimer benzimidazole conjugate 5 (DPA 83) was synthesized by conjugating a neomycin-dimer with a benzimidazole alkyne using click chemistry to target multiple binding sites on HIV TAR RNA. Ligand 5 significantly enhances the thermal stability of HIV TAR RNA and interacts stoichiometrically with HIV TAR RNA with a low nanomolar affinity. 5 displayed enhanced binding compared to its individual building blocks including the neomycin dimer azide and benzimidazole alkyne. In essence, a high affinity multivalent ligand was designed and synthesized to target HIV TAR RNA.

The nucleotides comprising the ribosomal decoding center are highly conserved, as they are important for maintaining translational fidelity. The bacterial A-site has a small base variation as compared with the human analogue, allowing aminoglycoside (AG) antibiotics to selectively bind within this region of the ribosome and negatively affect microbial protein synthesis. Here, by using a fluorescence displacement screening assay, we demonstrate that neomycin B (NEO) dimers connected by L-arginine-containing linkers of varying length and composition bind with higher affinity to model A-site RNAs compared to NEO, with IC50 values ranging from ~40–70 nM, and that a certain range of linker lengths demonstrates a clear preference for the bacterial A-site RNA over the human analogue. Furthermore, AG-modifying enzymes (AMEs), such as AG O-phosphotransferases, which are responsible for conferring antibiotic resistance in many types of infectious bacteria, demonstrate markedly reduced activity against several of the L-arginine-linked NEO dimers in vitro. The antimicrobial activity of these dimers against several bacterial strains is weaker than that of the parent NEO.

miRNAs are important components of regulatory networks that control gene expression and have implications in various diseases including cancer. Targeting oncogenic miRNAs with small molecules is currently being explored to develop cancer therapeutics. Here, we report the development of dual binding neomycin–bisbenzimidazole conjugates that target oncogenic miR-27a with high affinity (Ka = 1.2 to 7.4 × 108 M−1). These conjugates bring significant reduction (∼65% at 5 μM) in mature miRNA levels and penetrate easily in the cells where they localise both in the cytoplasm and the nucleus. Cell cycle analysis showed significant increase in the G0/G1 phase (∼15%) and decrease in the S phase (∼7%) upon treatment with neomycin–bisbenzimidazole conjugates, suggesting inhibition of cell proliferation. Using the conjugation approach, we show that moderately binding ligands can be covalently combined into high affinity binders. This study also highlights the role of linker optimization in designing high affinity ligands for miR-27a targeting.

A 215-member mono- and diamino acid peptidic-aminosugar (PA) library, with neomycin as the model aminosugar, was systematically and rapidly synthesized via solid phase synthesis. Antibacterial activities of the PA library, on 13 bacterial strains (seven Gram-positive and six Gram-negative bacterial strains), and binding affinities of the PA library for a 27-base model of the bacterial 16S ribosomal A-site RNA were evaluated using high-throughput screening. The results of the two assays were correlated using Ribosomal Binding-Bacterial Inhibition Plot (RB-BIP) analysis to provide structure–activity relationship (SAR) information. From this work, we have identified PAs that can discriminate the E. coli A-site from the human A-site by up to a 28-fold difference in binding affinity. Aminoglycoside-modifying enzyme activity studies indicate that APH(2″)-Ia showed nearly complete removal of activity with a number of PAs. The synthesis of the compound library and screening can both be performed rapidly, allowing for an iterative process of aminoglycoside synthesis and screening of PA libraries for optimal binding and antibacterial activity for lead identification.

DNA minor groove binding drugs such as Hoechst 33258 have been shown to bind to a number of RNA structures. Similarly, RNA binding ligands such as neomycin have been shown by us to bind to a number of A-form DNA structures. A neomycin–Hoechst 33258 conjugate was recently shown to bind B-DNA, where Hoechst exhibits high affinity for the minor groove of A/T tract DNA and neomycin docks into the major groove. Further studies now indicate that the Hoechst moiety of the conjugate can be driven to bind RNA duplex as a consequence of neomycin binding in the RNA major groove. This is the first example of Hoechst 33258 binding to RNA duplex not containing bulges or loop motifs.

Neomycin dimers synthesized using “click chemistry” with varying functionality and length in the linker region have been shown to be effective in targeting the HIV-1 transactivation response element (TAR) RNA region of the HIV virus. TAR, a 59 base pair stem loop structure located at the 5′-end of all nascent viral transcripts interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Ethidium bromide displacement and FRET competition assays have revealed nanomolar binding affinity between neomycin dimers and wildtype TAR RNA while in case of neomycin, only a weak binding was detected. Here, NMR and FID-based comparisons reveal an extended binding interface for neomycin dimers involving the upper stem of the TAR RNA thereby offering an explanation for increased affinities. To further explore the potential of these modified aminosugars we have extended binding studies to include four TAR RNA mutants that display conformational differences with minimal sequence variation. The differences in binding between neomycin and neomycin dimers is characterized with TAR RNA mutants that include mutations to the bulge region, hairpin region, and both the bulge and hairpin regions. Our results demonstrate the effect of these mutations on neomycin binding and our results show that linker functionalities between dimeric units of neomycin can distinguish between the conformational differences of mutant TAR RNA structures.

Hoechst dyes are well known DNA binders that non-selectively inhibit the function of mammalian topoisomerase I and II. Herein, we show that Hoechst 33258 based bisbenzimidazoles (DPA 151–154), containing a terminal alkyne, are effective and selective inhibitors of E. coli topoisomerase I. These bisbenzimidazoles displayed topoisomerase I inhibition much better than Hoechst 33342 or Hoechst 33258 with IC50 values in the range of 2.47–6.63 μM. Bisbenzimidazoles DPA 151–154 also display selective inhibition of E. coli topoisomerase I over DNA gyrase and human topoisomerases I and II, and effectively inhibit bacterial growth.

The authors report the recognition of a G-quadruplex formed by four repeat human telomeric DNA with aminosugar intercalator conjugates. The recognition of the G-quadruplex through dual binding mode ligands significantly increased the affinity of ligands for the G-quadruplex. One such example is a neomycin–anthraquinone conjugate (2) which exhibited nanomolar affinity for the quadruplex, and the affinity of (2) is nearly 1000 fold higher for the human telomeric G-quadruplex DNA than its constituent units, neomycin and anthraquinone.

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.

The development of new antibacterial agents has become necessary to treat the large number of emerging bacterial strains resistant to current antibiotics. Despite the different methods of resistance developed by these new strains, the A-site of the bacterial ribosome remains an attractive target for new antibiotics. To develop new drugs that target the ribosomal A-site, a high-throughput screen is necessary to identify compounds that bind to the target with high affinity. To this end, we present an assay that uses a novel fluorescein-conjugated neomycin (F-neo) molecule as a binding probe to determine the relative binding affinity of a drug library. We show here that the binding of F-neo to a model Escherichia coli ribosomal A-site results in a large decrease in the fluorescence of the molecule. Furthermore, we have determined that the change in fluorescence is due to the relative change in the pKa of the probe resulting from the change in the electrostatic environment that occurs when the probe is taken from the solvent and localized into the negative potential of the A-site major groove. Finally, we demonstrate that F-neo can be used in a robust, highly reproducible assay, determined by a Z′-factor greater than 0.80 for 3 consecutive days. The assay is capable of rapidly determining the relative binding affinity of a compound library in a 96-well plate format using a single channel electronic pipette. The current assay format will be easily adaptable to a high-throughput format with the use of a liquid handling robot for large drug libraries currently available and under development.

Covering: 1980 to 2011 Major groove recognition of DNA by proteins utilizes the variation in hydrogen bond donor/acceptor content that makes DNA base-pairs distinguishable from one another. Specific ligand–DNA interactions in the major groove are necessary to develop approaches for inhibition of DNA–protein interactions. As opposed to minor groove binders, little research has been achieved in recognition of the DNA major groove. This review summarizes the progress in identification of natural products that bind to the major groove of DNA. We first review the natural products, pluramycins, aflatoxins, azinomycins, leinamycin, neocarzinostatin, and ditercalinium, that are known to possess major groove interacting elements. These compounds, however, interact primarily with DNA by intercalation between base-pair steps. Some of these compounds utilize non-covalent interactions in order to position themselves to alkylate DNA at the nucleophilic N7 positions on nearby purine bases. Finally, recent reports of non-covalent major groove binding with carbohydrates, aminoglycosides in particular, have revealed them as promising leads for DNA major groove binding probes or drugs.

A series of neomycin dimers have been synthesized using “click chemistry” with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem–loop structure located at the 5′-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the melting temperature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC50 values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity.

Antisense strategies that target DNA·RNA hybrid structures offer potential for the development of new therapeutic drugs. The α-sarcin loop region of the 16S rRNA domain has been shown to be a high value target for such strategies. Herein, aminoglycoside interaction with three RNA·DNA α-sarcin targeted duplexes (rR·dY, rR·S-dY, and rR·2′OMe-rY) have been investigated to determine the overall effect of aminoglycoside interaction on the stability, affinity, and conformation of these hybrid duplexes. To this end, UV thermal denaturation, circular dichroism spectroscopy, fluorescence intercalator displacement, and ITC as well as DSC calorimetry experiments were carried out. The results suggest the following. (1) Of all the aminoglycosides studied, neomycin confers the highest thermal stability on all three hybrid duplexes studied. (2) There is no appreciable difference in aminoglycoside-induced thermal stability between the unmodified rR·dY and phophorothioate modified rR·S-dY duplexes. (3) The rR·2′OMe-rY duplexes thermal stability is slightly less than the other two hybrids. (4) In all three duplexes, aminoglycoside-induced thermal stability decreased as the number of amino groups decreased. (5) CD scans revealed similar spectra for the rR·dY and rR·S-dY duplexes as well as a more pronounced A-form signal for the rR·2′OMe-rY duplex. (6) FID assays paralleled the CD results, yielding similar affinity values between the rR·dY and rR·S-dY duplexes and higher affinities with the rR·2′OMe-rY duplex. (7) The overall affinity trend between aminoglycosides and the three duplexes was determined to be neomycin > paromomycin > neamine > ribostamycin. (8) ITC Ka values revealed similar binding constants for the rR·dY and rR·S-dY duplexes with rR·dY having a K1 of (1.03 ± 0.58) × 107 M–1 and K2 of (1.13 ± 0.07) × 105 M–1 while rR·S-dY produced a K1 of (1.17 ± 0.54) × 107 M–1 and K2 of (1.27 ± 0.69) × 105 M–1. (8) The rR·2′OMe-rY produced a slightly higher binding constant values with a K1 of (1.25 ± 0.24) × 107 M–1 and K2 of (3.62 ± 0.18) × 105 M–1. (9) The ΔTm-derived KTm of 3.81 × 107 M–1 for rR·S-dY was in relative agreement with the corresponding K1 of 1.17 × 107 M–1 derived constant from the fitted ITC. These results illustrate that the increased DNA·RNA hybrid duplex stability in the presence of aminoglycosides can help extend the roles of aminoglycosides in designing modified ODNs for targeting RNA.

A DNA duplex can be recognized sequence-specifically in the major groove by an oligodeoxynucleotide (ODN). The resulting structure is a DNA triple helix, or triplex. The scientific community has invested significant research capital in the study of DNA triplexes because of their robust potential for providing new applications, including molecular biology tools and therapeutic agents. The triplex structures have inherent instabilities, however, and the recognition of DNA triplexes by small molecules has been attempted as a means of strengthening the three-stranded complex. Over the decades, the majority of work in the field has focused on heterocycles that intercalate between the triplex bases. In this Account, we present an alternate approach to recognition and stabilization of DNA triplexes.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 TAT 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 (TAT 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 noncompeting 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.

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′]).

Synthesis of a novel perylene−neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5′-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA−RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV−vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand−quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

Recognition of nucleic acids is important for our understanding of nucleic acid structure as well as for our understanding of nucleic acid–protein interactions. In addition to the direct readout mechanisms of nucleic acids such as H-bonding, shape recognition of nucleic acids is being increasingly recognized as playing an equally important role in DNA recognition. Competition dialysis, UV, flourescent intercalator displacement (FID), computational docking, and calorimetry studies were conducted to study the interaction of neomycin with a variety of nucleic acid conformations (shapes). At pH 5.5, the results suggest the following. (1) Neomycin binds three RNA structures [16S A site rRNA, poly(rA)·poly(rA), and poly(rA)·poly(rU)] with high affinities (Ka ∼ 107 M–1). (2) The binding of neomycin to A-form GC-rich oligomer d(A2G15C15T2)2 has an affinity comparable to those of RNA structures. (3) The binding of neomycin to DNA·RNA hybrids shows a 3-fold variance that can be attributed to their structural differences [for poly(dA)·poly(rU), Ka = 9.4 × 106 M–1, and for poly(rA)·poly(dT), Ka = 3.1 × 106 M–1]. (4) The interaction of neomycin with DNA triplex poly(dA)·2poly(dT) yields a binding affinity (Ka) of 2.4 × 105 M–1. (5) Poly(dA-dT)2 shows the lowest association constant for all nucleic acids studied (Ka < 105). (6) Neomycin binds to G-quadruplexes with Ka values of ∼104–105 M–1. (7) Computational studies show that the decrease in major groove width in the B to A transition correlates with increasing neomycin affinity. Neomycin’s affinity for various nucleic acid structures can be ranked as follows: RNAs and GC-rich d(A2G15C15T2)2 structures > poly(dA)·poly(rU) > poly(rA)·poly(dT) > T·A-T triplex, G-quadruplex, B-form AT-rich, or GC-rich DNA sequences. The results illustrate the first example of a small molecule-based “shape readout” of different nucleic acid conformations.

A series of neomycin dimers have been synthesized using ‘click chemistry’ with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a 59 base pair stem loop structure located at 5′-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement titrations revealed nanomolar IC50 between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC50 in the micromolar range is observed.A series of neomycin dimers have been synthesized using ‘click chemistry’ to target the HIV virus TAR (trans activation response) RNA region. The neomycin dimers were synthesized in high yields and show a strong stabilizing effect on HIV TAR RNA, when compared to neomycin.

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 (Ka) ∼ 105 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.

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.

Thermodynamic studies on the interactions between intercalator−neomycin conjugates and a DNA polynucleotide triplex [poly(dA)·2poly(dT)] were conducted. To draw a complete picture of such interactions, naphthalene diimide−neomycin (3) and anthraquinone−neomycin (4) conjugates were synthesized and used together with two other analogues, previously synthesized pyrene−neomycin (1) and BQQ−neomycin (2) conjugates, in our investigations. A combination of experiments, including UV denaturation, circular dichroism (CD) titration, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC), revealed that all four conjugates (1−4) stabilized poly(dA)·2poly(dT) much more than its parent compound, neomycin. UV melting experiments clearly showed that the temperature (Tm3→2) at which poly(dA)·2poly(dT) dissociated into poly(dA)·poly(dT) and poly(dT) increased dramatically (>12 °C) in the presence of intercalator−neomycin conjugates (1−4) even at a very low concentration (2 μM). In contrast to intercalator−neomycin conjugates, the increment of Tm3→2 of poly(dA)·2poly(dT) induced by neomycin was negligible under the same conditions. The binding preference of intercalator−neomycin conjugates (1−4) to poly(dA)·2poly(dT) was also confirmed by competition dialysis and a fluorescent intercalator displacement assay. Circular dichroism titration studies revealed that compounds 1−4 had slightly larger binding site size (∼7−7.5) with poly(dA)·2poly(dT) as compared to neomycin (∼6.5). The thermodynamic parameters of these intercalator−neomycin conjugates with poly(dA)·2poly(dT) were derived from an integrated van’t Hoff equation using the Tm3→2 values, the binding site size numbers, and other parameters obtained from DSC and ITC. The binding affinity of all tested ligands with poly(dA)·2poly(dT) increased in the following order: neomycin < 1 < 3 < 4 < 2. Among them, the binding constant [(2.7 ± 0.3) × 108 M−1] of 2 with poly(dA)·2poly(dT) was the highest, almost 1000-fold greater than that of neomycin. The binding of compounds 1−4 with poly(dA)·2poly(dT) was mostly enthalpy-driven and gave negative ΔCp values. The results described here suggest that the binding affinity of intercalator−neomycin conjugates for poly(dA)·2poly(dT) increases as a function of the surface area of the intercalator moiety.

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.The synthesis and binding studies of a neomycin–Hoechst 33258-pyrene (NHP) conjugate capable of binding B-form DNA is presented. NHP raises the Tm of AT rich duplex DNA by 35 °C.

We report the synthesis of pyrene–neomycin conjugate and its ability to stabilize DNA/RNA triple helices.

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.

Nucleic acids adopt a broad array of hydrogen-bonded structures that enable their diverse roles in the cell; even the familiar DNA double helix displays subtle architectural nuances that are sequence dependent. While there have been many approaches for recognition of B-form nucleic acids, A-form DNA recognition has lagged behind. Here, using a tight binding fluorescein-neomycin (F-neo) conjugate that can probe the electrostatic environment of A-form DNA major groove, we developed a fluorescent displacement assay to be used as a screen for DNA duplex-binding compounds. As opposed to intercalating dyes that can significantly perturb DNA structure, the groove binding F-neo allows the probing of native DNA conformation. In combination with the assay development and probing of DNA grooves, we also report the synthesis and binding of a series of neomycin-anthraquinone conjugates, two units with a known preference for binding GC rich DNA. The assay can be used to identify duplex DNA-binding compounds, as well as probe structural features of a target DNA duplex, and can easily be scaled up for high throughput screening of compound libraries.

Neomycin and Hoechst 33258 are two well-known nucleic acid binders that interact with RNA and DNA duplexes with high affinities respectively. In this manuscript, we report that covalent attachment of bisbenzimidazole unit derived from Hoechst 33258 to neomycin leads to intercalative binding of the bisbenzimidazole unit (oriented at 64–74° with respected to the RNA helical axis) in a linker length dependent manner. The dual binding and intercalation of conjugates were supported by thermal denaturation, CD, LD and UV–Vis absorption experiments. These studies highlight the importance of linker length in dual recognition by conjugates, for effective RNA recognition, which can lead to novel ways of recognizing RNA structures. Additionally, the ligand library screens also identify DNA and RNA selective compounds, with compound 9, containing a long linker, showing a 20.3 °C change in RNA duplex Tm with only a 13.0 °C change in Tm for the corresponding DNA duplex. Significantly, the shorter linker in compound 3 shows almost the reverse trend, a 23.8 °C change in DNA Tm, with only a 9.1 °C change in Tm for the corresponding RNA duplex.

miRNAs are important components of regulatory networks that control gene expression and have implications in various diseases including cancer. Targeting oncogenic miRNAs with small molecules is currently being explored to develop cancer therapeutics. Here, we report the development of dual binding neomycin–bisbenzimidazole conjugates that target oncogenic miR-27a with high affinity (Ka = 1.2 to 7.4 × 108 M−1). These conjugates bring significant reduction (∼65% at 5 μM) in mature miRNA levels and penetrate easily in the cells where they localise both in the cytoplasm and the nucleus. Cell cycle analysis showed significant increase in the G0/G1 phase (∼15%) and decrease in the S phase (∼7%) upon treatment with neomycin–bisbenzimidazole conjugates, suggesting inhibition of cell proliferation. Using the conjugation approach, we show that moderately binding ligands can be covalently combined into high affinity binders. This study also highlights the role of linker optimization in designing high affinity ligands for miR-27a targeting.

The authors report the recognition of a G-quadruplex formed by four repeat human telomeric DNA with aminosugar intercalator conjugates. The recognition of the G-quadruplex through dual binding mode ligands significantly increased the affinity of ligands for the G-quadruplex. One such example is a neomycin–anthraquinone conjugate (2) which exhibited nanomolar affinity for the quadruplex, and the affinity of (2) is nearly 1000 fold higher for the human telomeric G-quadruplex DNA than its constituent units, neomycin and anthraquinone.

Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs. A novel neomycin dimer benzimidazole conjugate 5 (DPA 83) was synthesized by conjugating a neomycin-dimer with a benzimidazole alkyne using click chemistry to target multiple binding sites on HIV TAR RNA. Ligand 5 significantly enhances the thermal stability of HIV TAR RNA and interacts stoichiometrically with HIV TAR RNA with a low nanomolar affinity. 5 displayed enhanced binding compared to its individual building blocks including the neomycin dimer azide and benzimidazole alkyne. In essence, a high affinity multivalent ligand was designed and synthesized to target HIV TAR RNA.