RESUMO
Hfq is required by many Gram-negative bacteria to chaperone the interaction between small non-coding RNA (sRNA) and mRNA to facilitate annealing. Conversely and despite the presence of Hfq in many Gram-positive bacteria, sRNAs in Gram-positive bacteria bind the mRNA target independent of Hfq. Details provided by the Hfq structures from both Gram-negative and Gram-positive bacteria have demonstrated that despite a conserved global structure of the protein, variations of residues on the binding surfaces of Hfq results in the recognition of different RNA sequences as well as the ability of Hfq to facilitate the annealing of the sRNA to the mRNA target. Additionally, a subset of Gram-negative bacteria has an extended C-terminal Domain (CTD) that has been shown to affect the stability of the Hfq hexamer and increase the rate of release of the annealed sRNA-mRNA product. Here we review the structures of Hfq and biochemical data that have defined the interactions of the Gram-negative and Gram-positive homologues to highlight the similarities and differences in the interactions with RNA. These interactions provided a deeper understanding of the how Hfq functions to facilitate the annealing of sRNA-mRNA, the selectivity of the interactions with RNA, and the role of the CTD of Hfq in the interactions with sRNA.
Assuntos
Proteínas de Escherichia coli , Pequeno RNA não Traduzido , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Sequência de Bases , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Proteínas de Escherichia coli/genéticaRESUMO
Aminoglycoside functionalization as a tool for targeting natural and unnatural nucleic acids holds great promise in their development as diagnostic probes and medicinally relevant compounds. Simple synthetic procedures designed to easily and quickly manipulate amino sugar (neomycin, kanamycin) to more powerful and selective ligands are presented in this chapter. We describe representative procedures for (a) aminoglycoside conjugation and (b) preliminary screening for their nucleic acid binding and selectivity.
Assuntos
Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Canamicina/metabolismo , Neomicina/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Antibacterianos/química , Canamicina/química , Neomicina/químicaRESUMO
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.
Assuntos
DNA Forma A/análise , Fluoresceína/química , Corantes Fluorescentes/análise , Neomicina/química , Simulação de Acoplamento Molecular , Estrutura MolecularRESUMO
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.
Assuntos
Aminoglicosídeos/farmacologia , Benzimidazóis/farmacologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Aminoglicosídeos/síntese química , Aminoglicosídeos/química , Benzimidazóis/síntese química , Benzimidazóis/química , Sítios de Ligação/efeitos dos fármacos , Química Click , Relação Dose-Resposta a Droga , Ligantes , Conformação Molecular , Neomicina/química , Neomicina/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid.
Assuntos
Aminoglicosídeos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , MicroRNAs/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Aminoácidos/metabolismo , Sequência de Bases , Sítios de Ligação , Fluoresceína/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Dados de Sequência Molecular , Neomicina/química , Neomicina/farmacologia , Conformação de Ácido Nucleico , Ligação Proteica/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacosRESUMO
The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.
Assuntos
Antibacterianos/farmacologia , Neomicina/farmacologia , Ribossomos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Dimerização , Humanos , Testes de Sensibilidade Microbiana , Conformação Proteica , RNA Ribossômico/biossíntese , Relação Estrutura-Atividade , Tioureia/química , Triazóis/química , Ureia/químicaRESUMO
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(2â³)-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.
Assuntos
Amino Açúcares/química , Antibacterianos/farmacologia , Biblioteca de Peptídeos , RNA/metabolismo , Antibacterianos/metabolismo , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sequência de Carboidratos , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase activity and are potential cancer therapies. We have developed an assay for the screening of compounds with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluorescent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence displacement assay. The conjugation of the planar base stacking thiazole orange with the groove binding neomycin results in high affinity probe that can determine the relative binding affinity of high affinity HTG binding drugs in a high throughput format. The robust assay is applicable for the determination of the binding affinity of HTG in the presence of K(+) or Na(+).
Assuntos
Bioensaio/métodos , Quadruplex G , Benzotiazóis/química , Benzotiazóis/metabolismo , Humanos , Cinética , Ligantes , Neomicina/química , Neomicina/metabolismo , Quinolinas/química , Quinolinas/metabolismo , Espectrometria de Fluorescência , Telômero/metabolismoRESUMO
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.
Assuntos
Benzimidazóis/química , Neomicina/química , RNA Viral/metabolismo , Bisbenzimidazol/química , Bisbenzimidazol/metabolismo , Dicroísmo Circular , Repetição Terminal Longa de HIV , HIV-1/genética , Humanos , Ligantes , RNA Viral/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
We report here the affinity and antibacterial activity of a structurally similar class of neomycin dimers. The affinity of the dimer library for rRNA was established by using a screen that measures the displacement of fluorescein-neomycin (F-neo) probe from RNA. A rapid growth inhibition assay using a single drug concentration was used to examine the antibacterial activity. The structure-activity relationship data were then rapidly analyzed using a two-dimensional ribosomal binding-bacterial inhibition plot analysis.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Ribossomos/química , Enterobacter cloacae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Neomicina/química , Neomicina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Serratia marcescens/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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 pK(a) 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.
Assuntos
Antibacterianos/análise , Antibacterianos/metabolismo , Bioensaio/métodos , Ribossomos/metabolismo , Sítios de Ligação , Ligação Competitiva , Escherichia coli/metabolismo , Fluoresceína , Fluorescência , Ensaios de Triagem em Larga Escala/métodos , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The repressor of bacteriophage P22 (P22R) discriminates between its various DNA binding sites by sensing the identity of non-contacted base pairs at the center of its binding site. The "indirect readout" of these non-contacted bases is apparently based on DNA's sequence-dependent conformational preferences. The structures of P22R-DNA complexes indicate that the non-contacted base pairs at the center of the binding site are in the B' state. This finding suggests that indirect readout and therefore binding site discrimination depend on P22R's ability to either sense and/or impose the B' state on the non-contacted bases of its binding sites. We show here that the affinity of binding sites for P22R depends on the tendency of the central bases to assume the B'-DNA state. Furthermore, we identify functional groups in the minor groove of the non-contacted bases as the essential modulators of indirect readout by P22R. In P22R-DNA complexes, the negatively charged E44 and E48 residues are provocatively positioned near the negatively charged DNA phosphates of the non-contacted nucleotides. The close proximity of the negatively charged groups on protein and DNA suggests that electrostatics may play a key role in the indirect readout process. Changing either of two negatively charged residues to uncharged residues eliminates the ability of P22R to impose structural changes on DNA and to recognize non-contacted base sequence. These findings suggest that these negatively charged amino acids function to force the P22R-bound DNA into the B' state and therefore play a key role in indirect readout by P22R.
Assuntos
Bacteriófago P22/química , DNA Viral/química , Conformação de Ácido Nucleico , Proteínas Repressoras/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Bacteriófago P22/genética , Bacteriófago P22/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , DNA Viral/genética , DNA Viral/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Regiões Operadoras Genéticas , Ligação Proteica , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Eletricidade Estática , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Mg²âº shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²âº in the absence of free oxygen as a replacement for Mg²âº in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²âº in RNA folding and function can indeed be served by Fe²âº. The results of quantum mechanical calculations show that the geometry of coordination of Fe²âº by RNA phosphates is similar to that of Mg²âº. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²âº or Mg²âº. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²âº, and the hammerhead ribozyme are enhanced in the presence of Fe²âº compared to Mg²âº. All chemical footprinting and ribozyme assays in the presence of Fe²âº were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²âº. The combined biochemical and paleogeological data are consistent with a role for Fe²âº in an RNA World. RNA and Fe²âº could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²âº alone.
Assuntos
Ferro/metabolismo , Catálise , Magnésio/metabolismo , Conformação de Ácido Nucleico , RNA/química , RNA/genética , Dobramento de RNA/genética , RNA Catalítico/genética , Tetrahymena thermophila/genéticaRESUMO
The binding of proteins to specific sequences of DNA is an important feature of virtually all DNA transactions. Proteins recognize specific DNA sequences using both direct readout (sensing types and positions of DNA functional groups) and indirect readout (sensing DNA conformation and deformability). Previously we showed that the P22 c2 repressor N-terminal domain (P22R NTD) forces the central non-contacted 5'-ATAT-3' sequence of the DNA operator into the B' state, a state known to affect DNA hydration, rigidity and bending. Usually the B' state, with a narrow minor groove and a spine of hydration, is reserved for A-tract DNA (TpA steps disrupt A-tracts). Here, we have co-crystallized P22R NTD with an operator containing a central 5'-ACGT-3' sequence in the non-contacted region. C.G base pairs have not previously been observed in the B' state and are thought to prevent it. However, P22R NTD induces a narrow minor groove and a spine of hydration to 5'-ACGT-3'. We observe that C.G base pairs have distinctive destabilizing and disordering effects on the spine of hydration. It appears that the reduced stability of the spine results in a higher energy cost for the B to B' transition. The differential effect of DNA sequence on the barrier to this transition allows the protein to sense the non-contacted DNA sequence.
Assuntos
DNA Viral/química , DNA Viral/metabolismo , Conformação de Ácido Nucleico , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófago P22/genética , Bacteriófago P22/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , DNA Viral/genética , Substâncias Macromoleculares/química , Modelos Moleculares , Regiões Operadoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/genética , Eletricidade Estática , Termodinâmica , Valina/química , Proteínas Virais/genéticaRESUMO
Anomalous scattering is commonly used to solve X-ray structures. As discussed here, anomalous scattering is also useful for characterizing complex systems with mixed and partial occupancies, where true electron density is represented by unresolvable ensemble averages. The solvent environment surrounding nucleic acids is an example of such a system, as are some DNA-ligand systems. The atomic number and wavelength dependencies of anomalous scattering allow one to filter out the electron densities of C, N, and O, and to cleanly visualize the electron densities of heavier atoms. Therefore, anomalous scattering can make beacons of selected atoms. In addition, anomalous scattering provides a model-independent method for determining atomic identities. Here, we describe applications of anomalous scattering to the structure determination of DNA-platinum complexes and in cation associations of free DNA, of DNA-anthracycline complexes, of chemically modified DNA, and of DNA-protein complexes. The utility of Rb(+) and Tl(+) as K(+) substitutes is supported by similarities in Rb(+) and Tl(+) association with DNA.
Assuntos
DNA/química , DNA/metabolismo , Difração de Raios X/métodos , Antraciclinas/química , Antraciclinas/metabolismo , Sequência de Bases , Cristalografia por Raios X , DNA/genética , Metais/química , Metais/metabolismo , Modelos Moleculares , Conformação Molecular , Proteínas/química , Proteínas/metabolismo , TermodinâmicaRESUMO
The P22 c2 repressor protein (P22R) binds to DNA sequence-specifically and helps to direct the temperate lambdoid bacteriophage P22 to the lysogenic developmental pathway. We describe the 1.6 A X-ray structure of the N-terminal domain (NTD) of P22R in a complex with a DNA fragment containing the synthetic operator sequence [d(ATTTAAGATATCTTAAAT)]2. This operator has an A-T base pair at position 9L and a T-A base pair at position 9R and is termed DNA9T. Direct readout: nondirectional van der Waals interactions between protein and DNA appear to confer sequence-specificity. The structure of the P22R NTD-DNA9T complex suggests that sequence-specificity arises substantially from lock-and-key interaction of a valine with a complementary binding cleft on the major groove surface of DNA9T. The cleft is formed by four methyl groups on sequential base pairs of 5'-TTAA-3'. The valine cleft is intrinsic to the DNA sequence and does not arise from protein-induced DNA conformational changes. Protein-DNA hydrogen bonding plays a secondary role in specificity. Indirect readout: it is known that the noncontacted bases in the center of the complex are important determinants of affinity. The protein induces a transition of the noncontacted region from B-DNA to B'-DNA. The B' state is characterized by a narrow minor groove and a zigzag spine of hydration. The free energy of the transition from B- to B'-DNA is known to depend on the sequence. Thus, the observed DNA conformation and hydration allows for the formulation of a predictive model of the indirect readout phenomenon.
Assuntos
Técnicas Biossensoriais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Pareamento de Bases/fisiologia , Sequência de Bases , Sítios de Ligação , Técnicas Biossensoriais/métodos , Cristalografia por Raios X , DNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Água/farmacologiaRESUMO
Here, we describe the 1.6-A X-ray structure of the DDD (Dickerson-Drew dodecamer), which has been covalently modified by the tethering of four cationic charges. This modified version of the DDD, called here the DDD(4+), is composed of [d(CGCGAAXXCGCG)](2), where X is effectively a thymine residue linked at the 5 position to an n-propyl-amine. The structure was determined from crystals soaked with thallium(I), which has been broadly used as a mimic of K(+) in X-ray diffraction experiments aimed at determining positions of cations adjacent to nucleic acids. Three of the tethered cations are directed radially out from the DNA. The radially directed tethered cations do not appear to induce structural changes or to displace counterions. One of the tethered cations is directed in the 3' direction, toward a phosphate group near one end of the duplex. This tethered cation appears to interact electrostatically with the DNA. This interaction is accompanied by changes in helical parameters rise, roll, and twist and by a displacement of the backbone relative to a control oligonucleotide. In addition, these interactions appear to be associated with displacement of counterions from the major groove of the DNA.