Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins

RNA is an important target for drug discovery efforts. Several clinically used aminoglycoside antibiotics bind to bacterial rRNA and inhibit protein synthesis. Aminoglycosides, however, are losing efficacy due to their inherent toxicity and the increase in antibiotic resistance. Targeting of other RNAs is also becoming more attractive thanks to the discovery of new potential RNA drug targets through genome sequencing and biochemical efforts. Identification of new compounds that target RNA is therefore urgent, and we report here on the development of rapid screening methods to probe binding of low molecular weight ligands to proteins and RNAs. A series of aminoglycosides has been immobilized onto glass microscope slides, and binding to proteins and RNAs has been detected by fluorescence. Construction and analysis of the arrays is completed by standard DNA genechip technology. Binding of immobilized aminoglycosides to proteins that are models for study of aminoglycoside toxicity (DNA polymerase and phospholipase C), small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), and a large (approximately 400 nucleotide) group I ribozyme RNA is detected. The ability to screen large RNAs alleviates many complications associated with binding experiments that use isolated truncated regions from larger RNAs. These studies lay the foundation for rapid identification of small organic ligands from combinatorial libraries that exhibit strong and selective RNA binding while displaying decreased affinity to toxicity-causing proteins.     

NMR-based characterization of phenothiazines as a RNA binding scaffold

Phenothiazines were identified by virtual screening as promising ligands for HIV-1 TAR RNA and A-site ribosomal RNA, and binding in each case was verified experimentally. Consequently, since phenothiazines generally possess high bioavailability and low toxicity, we used several NMR techniques to explore the binding characteristics of acetopromazine with a total of five different RNA constructs: four as potential drug targets plus one control RNA construct. Acetopromazine was able to bind to various internal bulges and terminal loops containing both purines and pyrimidines, but no binding could be detected with double-stranded RNA or tetraloops. Dissociation constants determined via NMR varied from 0.27 to >3 mM. Analysis of differential saturation transfer difference (STD) NMR effects of acetopromazine suggests that the phenothiazine moiety has the closest contact to the binding sites of TAR and A-site RNA while the flexible N,N-dimethylpropylamino side chain contributes less to binding. NMR studies on A-site ribosomal RNA binding by six commercially available phenothiazines, while too few to establish a true structure-activity relationship, revealed a distinct dependence on aromatic ring and side chain substituents. Substituted phenothiazines have low molecular weight, are not highly charged, and have an inherent affinity for irregular tertiary RNA folds, suggesting that they can serve as a novel scaffold for constructing RNA-binding ligands.     

Specificity of aminoglycoside antibiotics for the A-site of the decoding region of ribosomal RNA

Background: Aminoglycoside antibiotics bind to the A-site of the decoding region of 16S RNA in the bacterial ribosome, an interaction that is probably responsible for their activity. A detailed study of the specificity of aminoglycoside binding to A-site RNA would improve our understanding of their mechanism of antibiotic activity.Results: We have studied the binding specificity of several aminoglycosides with model RNA sequences derived from the 16S ribosomal A-site using surface plasmon resonance. The 4,5-linked (neomycin) class of aminoglycosides showed specificity for wild-type A-site sequences, but the 4,6-linked class (kanamycins and gentamicins), generally showed poor specificity for the same sequences. Methylation of a cytidine in the target RNA, as found in the Escherichia coli ribosome, had negligible effects on aminoglycoside binding.Conclusions: Although both 4,5- and 4,6-linked aminoglycosides target the same ribosomal site, they appear to bind and effect antibiotic activity in different manners. The aminoglycosides might recognize different RNA conformations or the interaction might involve different RNA tertiary structures that are not equally sampled in our ribosome-free model. These results imply that models of ribosomal RNA must be carefully designed if the data are expected to accurately reflect biological activity.     

Stabilization of zwitterions in solution: GABA analogues

The solution-phase structures of a number of conformationally restricted gamma-aminobutyric acid (GABA) analogues are investigated at the MP2/6-31+G* level of theory, using both explicit water molecules and the conductor-like screening solvation model (COSMO) to model solvation. GABA analogues constrained in a cis conformation by either a double bond or cyclopropane ring have the potential to attain either folded, intramolecularly hydrogen-bonded, or partially folded conformations in solution. Systems constrained in a cis conformation by a cyclopentane or cyclopentene ring are more conformationally restricted and exist only in a folded, intramolecularly hydrogen-bonded form. GABA analogues constrained in a trans conformation by either a double bond or cyclopropane ring have the potential to adopt either partially folded or fully extended conformations in solution. Due to a lack of conformational flexibility, analogues that are constrained in a trans conformation by a cyclopentane or cyclopentene ring attain only partially folded conformations. Like GABA, conformationally flexible GABA analogues possess a large number of stable rotamers, and may exist in any or all of these conformations in aqueous solution. The structures of these analogues provide an essential foundation for subsequent structure-activity analysis of ligand binding at GABA receptors and transporters. This work is therefore expected to facilitate the design and development of new biologically active GABA analogues to treat GABA-related neurological disorders.     

Fluorescent HIV-1 Dimerization Initiation Site: design, properties, and use for ligand discovery

The HIV-1 Dimerization Initiation Site (DIS) is an intriguing, yet underutilized, viral RNA target for potential antiretroviral therapy. To study the recognition features of this target and to provide a quantitative, rapid, and real-time tool for the discovery of new binders, a fluorescence-based assay has been constructed. It relies on strategic incorporation of 2-aminopurine, an isosteric fluorescent adenosine analogue, into short hairpin RNA constructs. These oligomers self-associate to form a kissing loop that thermally rearranges into a more stable extended duplex, thereby mimicking the association and structural features of the native RNA sequence. We demonstrate the ability of two fluorescent DIS constructs, DIS272(2AP) and DIS273(2AP), to report the binding of known DIS binders via changes in their emission intensity. Binding of aminoglycosides such as paromomycin to DIS272(2AP) results in significant fluorescence enhancement, while ligand binding to DIS273(2AP) results in fluorescence quenching. These observations are rationalized by comparison to the sequence-analogous bacterial A-site, where the relative emission of the fluorescent probe is dependent on the placement of the flexible purine residues inside or outside the helical domain. Analysis of binding isotherms generated using DIS272(2AP) yields submicromolar EC50 values for paromomycin (0.5 +/- 0.2 microM) and neomycin B (0.6 +/- 0.2 microM). Other neomycin-family aminoglycosides are less potent binders with neamine, the core pharmacophore, displaying the lowest affinity of 21 +/- 1 microM. Screening of additional aminoglycosides and their derivatives led to the discovery of new, previously unreported, aminoglycoside binders of the HIV DIS RNA, among them butirosin A (5.5 +/- 0.6 microM) and apramycin (7.6 +/- 1.0 microM). A conformationally constrained neomycin B analogue displays a rather high affinity to the DIS (1.9 +/- 0.2 microM). Among a series of nucleobase aminoglycoside conjugates, only the uracil derivatives display a measurable affinity using this assay with EC50 values in the 2 microM range. In addition, similarity between the solution behavior of HIV-1 DIS and the bacterial decoding A-site has been observed, particularly with respect to the intra- and extra-helical residence of the conformationally flexible A residues within the bulge. Taken together, the observations reported here shed light on the solution behavior of this important RNA target and are likely to facilitate the design of new DIS selective ligands as potential antiretroviral agents.     

Structural mimicry of retroviral tat proteins by constrained beta-hairpin peptidomimetics: ligands with high affinity and selectivity for viral TAR RNA regulatory elements

An approach is described to the design of beta-hairpin peptidomimetic ligands for bovine immunodeficiency virus (BIV) Tat protein, which inhibit binding to its transactivator response element (TAR) RNA. A library of peptidomimetics was derived by grafting onto a hairpin-inducing d-Pro-l-Pro template sequences related to the RNA recognition element in Tat. One hairpin mimetic was identified that binds tightly (K(d) approximately 150 nM) to BIV TAR, and another that binds also to HIV-1 TAR RNA (K(d) approximately 1-2 microM). (In the same assay, the wild-type BIV Tat(65-81) peptide binds to BIV TAR with K(d) approximately 50 nM.) The high-affinity BIV-Tat mimetic was shown to adopt a stable beta-hairpin conformation in free solution by NMR methods. Amino acid substitutions in this mimetic were shown to impact on the hairpin structure and to disrupt binding to the RNA. This family of conformationally constrained peptidomimetics affords insights into the structural requirements for binding to TAR RNA and provides a basis for the design of new ligands with increased inhibitory activity and specificity to both BIV and HIV TAR RNAs.     

Scanning conformational space with a library of stereo- and regiochemically diverse aminoglycoside derivatives: the discovery of new ligands for RNA hairpin sequences

A library of stereo- and regiochemically diverse aminoglycoside derivatives was screened at 1 microM using surface plasmon resonance (SPR) against RNA hairpin models of the bacterial A-site, and the HIV viral TAR and RRE sequences. In order to double the stereochemical diversity of the library, the compounds were screened against both enantiomers of each of these sequences. Remarkably, this initial screen suggested that the same four aminoglycoside derivatives bound most tightly to all three of the RNAs, suggesting that these compounds were good RNA binders which, nonetheless, discriminated poorly between the RNA sequences. The interactions between selected isomeric aminoglycoside derivatives and the RNA hairpins were then studied in more detail using an SPR assay. Three isomeric tight-binding aminoglycoside derivatives, which had been identified from the initial screen, were found to bind more tightly to the RNA hairpins (with K(D) values in the range 0.23 to 4.7 microM) than a fourth isomeric derivative (which had K(D) values in the range 6.0 to 30 microM). The magnitude of the tightest RNA-aminoglycoside interactions stemmed, in large part, from remarkably slow dissociation of the aminoglycosides from the RNA targets. The three tight-binding aminoglycoside derivatives were found, however, to discriminate rather poorly between alternative RNA sequences with, at best, around a twenty-fold difference in affinity for alternative RNA hairpin sequences. Within the aminoglycoside derivative library studied, high affinity for an RNA target was not accompanied by good discrimination between alternative RNA sequences.     

Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

The recognition of the aminoglycosides neomycin and streptomycin by HIV-1 TAR RNA was studied by electrospray ionization mass spectrometry (ESI-MS). Members of the aminoglycoside family of antibiotics are known to target a wide variety of RNA molecules. Neomycin and streptomycin inhibit the formation of the Tat protein–TAR RNA complex, an assembly that is believed to be necessary for HIV replication. The noncovalent complexes formed by the binding of aminoglycosides to TAR RNA and the Tat–TAR complex were detected by ESI-MS. Neomycin has a maximum binding stoichiometry of three and two to TAR RNA and to the Tat–TAR complex, respectively. Data from the ESI-MS experiments suggest that a high affinity binding site of neomycin is located near the three-nucleotide bulge region of TAR RNA. This is consistent with previous solution phase footprinting measurements [H.-Y. Mei et al., Biochemistry 37 (1998) 14204]. Neomycin has a higher affinity toward TAR RNA than streptomycin, as measured by ESI-MS competition binding experiments. A noncovalent complex formed between a small molecule inhibitor of TAR RNA, which has a similar solution binding affinity as the aminoglycosides, and TAR RNA is much less stable than the RNA–aminoglycoside complexes to collisional dissociation in the gas phase. It is believed that the small molecule inhibitor interacts with TAR RNA via hydrophobic interactions, whereas the aminoglycosides bind to RNAs through electrostatic forces. This difference in gas phase stabilities may prove useful for discerning the types of noncovalent forces holding complexes together.     

Calorimetric and structural studies of 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide inhibitors of Src SH2 domain binding.

Isothermal titration calorimetry and X-ray crystallography have been used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEEI ligand for the Src Homology 2 domain of the Src kinase (Src SH2 domain). The conformationally constrained peptide mimics that were used are cyclopropane-derived isosteres whereby a cyclopropane ring substitutes to the N-Calpha-Cbeta atoms of the phosphotyrosine. Comparison of the thermodynamic data for the binding of the conformationally constrained peptide mimics relative to their equivalent flexible analogues as well as a native tetrapeptide revealed an entropic advantage of 5-9 cal mol(-1) K(-1) for the binding of the conformationally constrained ligands. However, an unexpected drop in enthalpy for the binding of the conformationally constrained ligands relative to their flexible analogues was also observed. To evaluate whether these differences reflected conformational variations in peptide binding modes, we have determined the crystal structure of a complex of the Src SH2 domain bound to one of the conformationally constrained peptide mimics. Comparison of this new structure with that of the Src SH2 domain bound to a natural 11-mer peptide (Waksman et al. Cell 1993, 72, 779-790) revealed only very small differences. Hence, cyclopropane-derived peptides are excellent mimics of the bound state of their flexible analogues. However, a rigorous analysis of the structures and of the surface areas at the binding interface, and subsequent computational derivation of the energetic binding parameters, failed to predict the observed differences between the binding thermodynamics of the rigidified and flexible ligands, suggesting that the drop in enthalpy observed with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remains unclear.     

Target-Directed Azide-Alkyne Cycloaddition for Assembling HIV-1 TAR RNA Binding Ligands

The highly conserved HIV-1 transactivation response element (TAR) binds to the trans-activator protein Tat and facilitates viral replication in its latent state. The inhibition of Tat–TAR interactions by selectively targeting TAR RNA has been used as a strategy to develop potent antiviral agents. Therefore, HIV-1 TAR RNA represents a paradigmatic system for therapeutic intervention. Herein, we have employed biotin-tagged TAR RNA to assemble its own ligands from a pool of reactive azide and alkyne building blocks. To identify the binding sites and selectivity of the ligands, the in situ cycloaddition has been further performed using control nucleotide (TAR DNA and TAR RNA without bulge) templates. The hit triazole-linked thiazole peptidomimetic products have been isolated from the biotin-tagged target templates using streptavidin beads. The major triazole lead generated by the TAR RNA presumably binds in the bulge region, shows specificity for TAR RNA over TAR DNA, and inhibits Tat–TAR interactions.     

Discovery of aminoglycoside mimetics by NMR-based screening of Escherichia coli A-site RNA

A method is described for the NMR-based screening for the discovery of aminoglycoside mimetics that bind to Escherichia coli A-site RNA. Although aminoglycosides are clinically useful, they exhibit high nephrotoxicity and ototoxicity, and their overuse has led to the development of resistance to important microbial pathogens. To identify a new series of aminoglycoside mimetics that could potentially overcome the problems associated with toxicities and resistance development observed with the aminoglycosides, we have prepared large quantities of E. coli 16 S A-site RNA and conducted an NMR-based screening of our compound library in search for small-molecule RNA binders against this RNA target. From these studies, several classes of compounds were identified as initial hits with binding affinities in the range of 70 microM to 3 mM. Lead optimization through synthetic modifications of these initial hits led to the discovery of several small-molecule aminoglycoside mimetics that are structurally very different from the known aminoglycosides. Structural models of the A-site RNA/ligand complexes were prepared and compared to the three-dimensional structures of the RNA/aminoglycoside complexes.     

Aminoglycoside Conjugation for RNA Targeting: Antimicrobials and Beyond

Natural aminoglycosides are therapeutically useful antibiotics and very efficient RNA ligands. They are oligosaccharides that contain several ammonium groups able to interfere with the translation process in prokaryotes upon binding to bacterial ribosomal RNA (rRNA), and thus, impairing protein synthesis. Even if aminoglycosides are commonly used in therapy, these RNA binders lack selectivity and are able to bind to a wide number of RNA sequences/structures. This is one of the reasons for their toxicity and limited applications in therapy. At the same time, the ability of aminoglycosides to bind to various RNAs renders them a great source of inspiration for the synthesis of new binders with improved affinity and specificity toward several therapeutically relevant RNA targets. Thus, a number of studies have been performed on these complex and highly functionalized compounds, leading to the development of various synthetic methodologies toward the synthesis of conjugated aminoglycosides. The aim of this review is to highlight recent progress in the field of aminoglycoside conjugation, paying particular attention to modifications performed toward the improvement of affinity and especially to the selectivity of the resulting compounds. This will help readers to understand how to introduce a desired chemical modification for future developments of RNA ligands as antibiotics, antiviral, and anticancer compounds.     

Target‐Directed Azide‐Alkyne Cycloaddition for Assembling HIV‐1 TAR RNA Binding Ligands

The highly conserved HIV‐1 transactivation response element (TAR) binds to the trans‐activator protein Tat and facilitates viral replication in its latent state. The inhibition of Tat–TAR interactions by selectively targeting TAR RNA has been used as a strategy to develop potent antiviral agents. Therefore, HIV‐1 TAR RNA represents a paradigmatic system for therapeutic intervention. Herein, we have employed biotin‐tagged TAR RNA to assemble its own ligands from a pool of reactive azide and alkyne building blocks. To identify the binding sites and selectivity of the ligands, the in situ cycloaddition has been further performed using control nucleotide (TAR DNA and TAR RNA without bulge) templates. The hit triazole‐linked thiazole peptidomimetic products have been isolated from the biotin‐tagged target templates using streptavidin beads. The major triazole lead generated by the TAR RNA presumably binds in the bulge region, shows specificity for TAR RNA over TAR DNA, and inhibits Tat–TAR interactions.     

Conformationally constrained analogues of bleomycin A5

The bleomycin (BLM) group antitumor antibiotics are glycopeptide-derived natural products shown to cause sequence selective lesions in DNA. Prior studies have indicated that the linker region, composed of the methylvalerate and threonine residues, may be responsible for a conformational bend in the agent required for efficient DNA cleavage. We have synthesized a number of conformationally constrained methylvalerate analogues and incorporated them into deglycobleomycin A(5) congeners using our recently reported procedure for the solid phase construction of (deglyco)bleomycin and its analogues. These analogues were designed to probe the effects of conformational constraint of the native valerate moiety. Initial experiments indicated that the constrained molecules, none of which mimic the conformation proposed for the natural valerate linker, possessed DNA cleavage activity, albeit with potencies less than that of (deglyco)BLM and lacking sequence selectivity. Further experiments demonstrated that these analogues failed to produce alkali-labile lesions in DNA or sequence selective oxidative damage in RNA. However, two of the conformationally constrained deglycoBLM analogues were shown to mediate RNA cleavage in the absence of added Fe(2+). The ability of the analogues to mediate the oxygenation of small molecules was also assayed, and it was shown that they were as competent in the transfer of oxygen to low molecular weight substrates as the parent compound.     

MCSS-based predictions of RNA binding sites

The diversity of RNA tertiary structures provides the basis for specific recognition by proteins or small molecules. To investigate the structural basis and the energetics which control RNA-ligand interactions, favorable RNA binding sites are identified using the MCSS method, which has been employed previously only for protein receptors. Two different RNAs for which the structures have been determined by NMR spectroscopy were examined: two structures of the TAR RNA which contains an arginine binding site, and the structure of the 16S rRNA which contains an aminoglycoside binding site (paromomycin). In accord with the MCSS methodology, the functional groups representing the entire ligand or only part of it (one residue in the case of the aminoglycosides) are first replicated and distributed with random positions and orientations around the target and then energy minimized in the force field of the target RNA. The Coulombic term and the dielectric constant of the force field are adjusted to approximate the effects of solvent-screening and counterions. Optimal force field parameters are determined to reproduce the binding mode of arginine to the TAR RNA. The more favorable binding sites for each residue of the aminoglycoside ligands are then calculated and compared with the binding sites observed experimentally. The predictability of the method is evaluated and refinements are proposed to improve its accuracy. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 7 December 1998     

The pattern of distribution of amino groups modulates the structure and dynamics of natural aminoglycosides: implications for RNA recognition

Aminoglycosides are clinically relevant antibiotics that participate in a large variety of molecular recognition processes involving different RNA and protein receptors. The 3-D structures of these policationic oligosaccharides play a key role in RNA binding and therefore determine their biological activity. Herein, we show that the particular NH2/NH3(+)/OH distribution within the antibiotic scaffold modulates the oligosaccharide conformation and flexibility. In particular, those polar groups flanking the glycosidic linkages have a significant influence on the antibiotic structure. A careful NMR/theoretical analysis of different natural aminoglycosides, their fragments, and synthetic derivatives proves that both hydrogen bonding and charge-charge repulsive interactions are at the origin of this effect. Current strategies to obtain new aminoglycoside derivatives are mainly focused on the optimization of the direct ligand/receptor contacts. Our results strongly suggest that the particular location of the NH2/NH3(+)/OH groups within the antibiotics can also modulate their RNA binding properties by affecting the conformational preferences and inherent flexibility of these drugs. This fact should also be carefully considered in the design of new antibiotics with improved activity.     

Binding affinity and inhibitory potency of neomycin and streptomycin on the Tat peptide interaction with HIV-1 TAR RNA detected by on-line acoustic wave sensor

The binding of two aminoglycoside antibiotics, neomycin and streptomycin, to a segment of the transactivation responsive region (TAR) RNA of the human immunodeficiency virus, and their inhibitory potency to disrupt the interaction of the RNA with a regulatory Tat protein-derived peptide, have been studied using a flow-through acoustic wave detector system. Binding affinity is directly correlated with the inhibitory potency of these molecules and the acoustic wave detection system shows that neomycin exhibits at least a ten-fold greater affinity for TAR RNA and that it is also a more potent inhibitor than streptomycin. These results are in agreement with previous studies. However, unlike the time-consuming batch-based assays, use of the flow-through format offers considerable potential for the rapid screening of the chemistry of relatively small-molecule-nucleic acid binding events.     

Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences

Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.     

Molecular dynamics study of the ribosomal A-site

Many aminoglycosidic antibiotics target the A-site of 16S RNA in the small ribosomal subunit and affect the fidelity of protein translation in bacteria. Upon binding, aminoglycosides displace two adenines (A1492 and A1493 for E. coli numbering) that are involved in tRNA anticodon loop recognition. The major difference in the aminoglycosidic binding site between the prokaryota and eukaryota is an adenine into guanine substitution in the position 1408. This mutation likely affects the dynamics of near A1492 and A1493 and hinders the binding of aminoglycosides to eukaryotic ribosomes. With multiple 20 ns long all-atom molecular dynamics simulations, we study the flexibility of a 22 nucleotide RNA fragment which mimics the aminoglycosidic binding site. Simulations are carried out for both native and A1408G mutated RNA as well as for their complexes with aminoglycosidic representative paromomycin. We observe intra- and extrahelical configurations of A1492 and A1493, which differ between the prokaryotic and the mutated structure. We obtain configurations of the A-site that are also observed in the NMR and crystal structures. Our studies show the differences in the internal mobility of the A-site, as well as that in ion and water density distributions inside of the binding cleft, between the prokaryotic and mutated RNA. We also compare the performance of two force field parameters for RNA, Amber and Charmm.