Specificity in the binding of aminoglycosides to HIV-RRE RNA.

Quantitative studies of the binding of neomycin B to RRE constructs are carried out to determine the relationship between non-Watson Crick base-paired elements in the RNA and aminoglycoside binding. The RRE region contains two unpaired domains containing a single base bulge and a bubble structure, respectively. Deletion of the single base bulge has no effect on neomycin binding as the site of aminoglycoside binding is localized to the bubble region. Converting the bubble region into an A-form duplex gradually abolishes neomycin B binding in 3-5-fold steps in affinity over a 75-fold range. Thus, the binding of aminoglycoside is favored at domains in RNA that are nonduplex in nature, but aminoglycoside binding is only graded-specific in that affinities are enhanced gradually as the structure further deviates from a duplex form. It is likely that high-affinity aminoglycoside binding does not occur in duplex RNA because the major groove is too narrow to allow for aminoglycoside access and that structural perturbations that allow widening of the groove facilitate access. However, these interactions are only graded-specific with respect to both aminoglycoside structure and RNA domain structure.     

Thermodynamics of aminoglycoside-rRNA recognition: the binding of neomycin-class aminoglycosides to the A site of 16S rRNA

We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA. Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration. (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups. (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH. Specifically, increasing the pH results in a corresponding increase in the number of linked protons. (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH. (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration. In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH. Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases. (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycin > paromomycin > ribostamycin. The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin. (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA. In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide. Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.     

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 K_d 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.     

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element

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) 相似文献   

Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at the 5'-phosphate.

The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5'-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotide was effected via condensation of the 2-cyanoethyl N,N-diisopropylphosphoramidite of 4-(1-pyrenyl)butanol (pyr-m4OPAm, 2) with the 5'-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The properties of the 5'-(pyr-m4)d(T)15 (3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3% relative to an identical concentration of free 4-(1-pyrenyl)butanol. The ultraviolet spectrum of the 5'-(pyr-m4)-d(T)15 and 5'-(pyr-m4)-d(T)15-d-(A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(1-pyrenyl)-butanol. The Tm values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9 degrees C, respectively. The pyrene-induced stabilization corresponds to a free energy change (delta delta G degrees) of -2.6 kcal/mol.     

Synthesis and antisense properties of oligodeoxyribonucleotides containing C5-substituted arabinofuranosyluracil

An oligodeoxyribonucleotide (ODN) containing three C5-substituted arabinofuranosyluracils was synthesized by the post-synthetic modification method from the ODN containing three C5-substituted 2,2'-anhydrouridines. The stability of the modified ODN/DNA duplex was lower than that of the corresponding normal duplex but that of the modified ODN/RNA duplex showed little change. The modified ODN could induce RNase H activity and was resistant against nuclease.     

Synthesis of aminoglycoside-3'-conjugates of 2'-O-methyl oligoribonucleotides and their invasion to a 19F labeled HIV-1 TAR model

The potential of aminoglycosides to induce RNA-invasion has been demonstrated. For this purpose, aminoglycoside-3'-conjugates of 2'-O-methyl oligoribonucleotides have been synthesized entirely on a solid phase. The synthesis includes an automated oligonucleotide chain elongation to solid-supported neomycin, ribostamycin, and methyl neobiosamine, and a two-step deprotection/release of the solid-supported conjugate, which allows exploitation of a simple protecting group scheme. Conjugates have been targeted to a (19)F labeled HIV-1 TAR RNA model (Trans Activation Response element of HIV), which allows monitoring of the invasion by (19)F NMR spectroscopy. A remarkably enhanced invasion, compared to that resulting from the corresponding unmodified 2'-O-methyl oligoribonucleotide (5'-CAGGCUCA-3'), has been obtained by the neomycin conjugate. The increased affinity results from a cooperative binding of the neomycin moiety and hybridization, though the invasion may also follow a mechanism, in which the first molar equivalent of the conjugate induces hybridization of the second.     

Synthesis and antibacterial activity of amphiphilic lysine-ligated neomycin B conjugates

Amphiphilic lysine-ligated neomycin B building blocks were prepared by reductive amination of a protected C5″-modified neomycin B-based aldehyde and side chain-unprotected lysine or lysine-containing peptides. It was demonstrated that a suitably protected lysine-ligated neomycin B conjugate (NeoK) serves as a building block for peptide synthesis, enabling incorporation of aminoglycoside binding sites into peptides. Antibacterial testing of three amphiphilic lysine-ligated neomycin B conjugates against a representative panel of Gram-positive and Gram-negative strains demonstrates that C5″-modified neomycin-lysine conjugate retains antibacterial activity. However, in most cases the lysine-ligated neomycin B analogs display reduced potency against Gram-positive strains when compared to unmodified neomycin B or unligated peptide. An exception is MRSA where an eightfold enhancement was observed. When compared to unmodified neomycin B, the prepared lysine-neomycin conjugates exhibited a 4–8-fold enhanced Gram-negative activity against Pseudomonas aeruginosa and up to 12-fold enhanced activity was observed when compared to unligated reference peptides.     

Efficient Stabilization of Phosphodiester (PO), Phosphorothioate (PS), and 2'-O-Methoxy (2'-OMe) DNA·RNA Hybrid Duplexes by Amino Sugars

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 K(a) values revealed similar binding constants for the rR·dY and rR·S-dY duplexes with rR·dY having a K(1) of (1.03 ± 0.58) × 10(7) M(-1) and K(2) of (1.13 ± 0.07) × 10(5) M(-1) while rR·S-dY produced a K(1) of (1.17 ± 0.54) × 10(7) M(-1) and K(2) of (1.27 ± 0.69) × 10(5) M(-1). (8) The rR·2'OMe-rY produced a slightly higher binding constant values with a K(1) of (1.25 ± 0.24) × 10(7) M(-1) and K(2) of (3.62 ± 0.18) × 10(5) M(-1). (9) The ΔT(m)-derived K(Tm) of 3.81 × 10(7) M(-1) for rR·S-dY was in relative agreement with the corresponding K(1) of 1.17 × 10(7) 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.     

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.     

Oligonucleotide conjugated to linear and branched high molecular weight polyethylene glycol as substrates for RNase H

Two conjugates of an anti-HIV oligonucleotide (ODN) with different high molecular weight monomethoxy polyethylene glycols (MPEGs) have been tested for their activity as substrate towards RNase H. The MPEG does not impede the formation of the regular hybrid duplex with the target RNA sequence as pointed out by the persistence of the RNase H activity; thus, these derivatives stimulate the hydrolysis of RNA by the enzyme at the same site and with the same extent of cleavage as the native sequence.     

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).     

Parameters of binding and transport of oligodeoxynucleotide, which includes BCL2 mRNA translation start, to the K562 cells

Interaction of oligodeoxynucleotides (ODN), 18-mer, which included sequence of BCL2 mRNA translation start, with K562 cells has been studied. The kinetic curves of interaction showed that oligonucleotide total binding with the cells at 37 degrees C and low oligonucleotide concentration (< or = 30 nM), as well as under lipofection, were composed of two processes: 18-mer surface binding with cell membranes and its non-proportional internalization into the cells. The last, in turn, consisted of three consequent steps. The enhanced extent and rate of oligonucleotide internalization was diminished after first hour incubation and later they were increased again. This reflected rising additional binding sites that provided internalization. At chosen time-points the internalization of ODN into cells, been proceeded at 37 degrees C, were at most abruptly abrogated by cooling down. ODN to K562 cell membrane binding constants and specific number of binding sites have been determined. Time-intervals, providing equilibrium for each successive stage of multistep ODN bound/free determination, were maintained. It was established that receptor binding with increased binding constant (more than 2 x 10(9) M(-1)) promoted ODN internalization. Oligonucleotide binding and internalization with prolonged incubation were also up-regulated due to priming new binding sites of higher affinity. Lipofection enhanced ODN binding to cell membrane but conserved the main features of ligand-receptor interaction. During lipofection constants and ODN binding site numbers increased without changing the overall time-pattern of the process, observed for ODN without liposomes. Extent and rate of internalization of ODN in liposomal formulation did not differ substantially from ODN in solution.     

Coupling of drug protonation to the specific binding of aminoglycosides to the A site of 16 S rRNA: elucidation of the number of drug amino groups involved and their identities

2-Deoxystreptamine (2-DOS) aminoglycoside antibiotics bind specifically to the central region of the 16S rRNA A site and interfere with protein synthesis. Recently, we have shown that the binding of 2-DOS aminoglycosides to an A site model RNA oligonucleotide is linked to the protonation of drug amino groups. Here, we extend these studies to define the number of amino groups involved as well as their identities. Specifically, we use pH-dependent 15N NMR spectroscopy to determine the pK(a) values of the amino groups in neomycin B, paromomycin I, and lividomycin A sulfate, with the resulting pK(a) values ranging from 6.92 to 9.51. For each drug, the 3-amino group was associated with the lowest pK(a), with this value being 6.92 in neomycin B, 7.07 in paromomycin I, and 7.24 in lividomycin A. In addition, we use buffer-dependent isothermal titration calorimetry (ITC) to determine the number of protons linked to the complexation of the three drugs with the A site model RNA oligomer at pH 5.5, 8.8, or 9.0. At pH 5.5, the binding of the three drugs to the host RNA is independent of drug protonation effects. By contrast, at pH 9.0, the RNA binding of paromomycin I and neomycin B is coupled to the uptake of 3.25 and 3.80 protons, respectively, with the RNA binding of lividomycin A at pH 8.8 being coupled to the uptake of 3.25 protons. A comparison of these values with the protonation states of the drugs predicted by our NMR-derived pK(a) values allows us to identify the specific drug amino groups whose protonation is linked to complexation with the host RNA. These determinations reveal that the binding of lividomycin A to the host RNA is coupled to the protonation of all five of its amino groups, with the RNA binding of paromomycin I and neomycin B being linked to the protonation of four and at least five amino groups, respectively. For paromomycin I, the protonation reactions involve the 1-, 3-, 2'-, and 2"'-amino groups, while, for neomycin B, the binding-linked protonation reactions involve at least the 1-, 3-, 2', 6'-, and 2"'-amino groups. Our results clearly identify drug protonation reactions as important thermodynamic participants in the specific binding of 2-DOS aminoglycosides to the A site of 16S rRNA.     

Aminoglycoside binding displaces a divalent metal ion in a tRNA-neomycin B complex

Aminoglycosides bind to RNA and interfere with its function, and it has been suggested that aminoglycoside binding to RNA displaces essential divalent metal ions. Here we demonstrate that addition of various aminoglycosides inhibited Pb2+-induced cleavage of yeast tRNA(Phe). Cocrystallization of yeast tRNA(Phe) and an aminoglycoside, neomycin B, resulted in crystals that diffracted to 2.6 A and the structure of the complex was solved by molecular replacement. The structure shows that the neomycin B binding site overlaps with known divalent metal ion binding sites in yeast tRNA(Phe), providing direct evidence for the hypothesis that aminoglycosides displace metal ions. Additionally, the neomycin B binding site overlaps with major determinants for Escherichia coli phenylalanyl-tRNA-synthetase. Here we present data demonstrating that addition of neomycin B inhibited aminoacylation of E. coli tRNA(Phe) in the mid microM range. Given that aminoglycoside and metal ion binding sites overlap, we discuss that aminoglycosides can be considered as 'metal mimics'.     

Quantitative affinity electrophoresis of RNA–small molecule interactions by cross-linking the ligand to acrylamide

We show that the affinity electrophoresis analysis of RNA–small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA–aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide–aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C–C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand.     

Recognition of RNA duplex by a neomycin–Hoechst 33258 conjugate

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.     

Recognition of B-DNA by neomycin--Hoechst 33258 conjugates

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.     

Neomycin induces high-affinity agonist binding of G-protein-coupled receptors

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.     

Synthesis and hybridization property of an oligonucleotide containing a 3-thioformacetal linked pentathymidylate

A pentathymidylate fully substituted with 3'-thioformacetal intemucleotidic linkages was synthesized and subsequently incorporated into an oligonucleotide (ON) 15mer. Tm analysis was performed on the resulting ON hybridized with its complementary RNA. This duplex demonstrated slightly improved binding affinity relative to the control phosphate diester ON/RNA hybrid.