Measurement of the specific and non-specific binding energies of Mg2+ to RNA

Determining the non-specific and specific electrostatic contributions of magnesium binding to RNA is a challenging problem. We introduce a single-molecule method based on measuring the folding energy of a native RNA in magnesium and at its equivalent sodium concentration. The latter is defined so that the folding energy in sodium equals the non-specific electrostatic contribution in magnesium. The sodium equivalent can be estimated according to the empirical 100/1 rule (1 M NaCl is equivalent to 10 mM MgCl₂), which is a good approximation for most RNAs. The method is applied to an RNA three-way junction (3WJ) that contains specific Mg²⁺ binding sites and misfolds into a double hairpin structure without binding sites. We mechanically pull the RNA with optical tweezers and use fluctuation theorems to determine the folding energies of the native and misfolded structures in magnesium (10 mM MgCl₂) and at the equivalent sodium condition (1 M NaCl). While the free energies of the misfolded structure are equal in magnesium and sodium, they are not for the native structure, the difference being due to the specific binding energy of magnesium to the 3WJ, which equals $\text{Δ}G?$ 10 kcal/mol. Besides stabilizing the 3WJ, Mg²⁺ also kinetically rescues it from the misfolded structure over timescales of tens of seconds in a force-dependent manner. The method should generally be applicable to determine the specific binding energies of divalent cations to other tertiary RNAs.     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.     

Surprising roles of electrostatic interactions in DNA-ligand complexes

The positions of cations in x-ray structures are modulated by sequence, conformation, and ligand interactions. The goal here is to use x-ray diffraction to help resolve structural and thermodynamic roles of specifically localized cations in DNA-anthracycline complexes. We describe a 1.34 A resolution structure of a CGATCG(2)-adriamycin(2) complex obtained from crystals grown in the presence of thallium (I) ions. Tl(+) can substitute for biological monovalent cations, but is readily detected by distinctive x-ray scattering, obviating analysis of subtle differences in coordination geometry and x-ray scattering of water, sodium, potassium, and ammonium. Six localized Tl(+) sites are observable adjacent to each CGATCG(2)-adriamycin(2) complex. Each of these localized monovalent cations are found within the G-tract major groove of the intercalated DNA-drug complex. Adriamycin appears to be designed by nature to interact favorably with the electrostatic landscape of DNA, and to conserve the distribution of localized cationic charge. Localized inorganic cations in the major groove are conserved upon binding of adriamycin. In the minor groove, inorganic cations are substituted by a cationic functional group of adriamycin. This partitioning of cationic charge by adriamycin into the major groove of CG base pairs and the minor groove of AT base pairs may be a general feature of sequence-specific DNA-small molecule interactions and a potentially useful important factor in ligand design.     

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA. I. Thermal unfolding pattern of lupin seeds and wheat germ 5S rRNAs, also in the presence of magnesium and sperminium cations.

An attempt has been made to correlate differential scanning calorimetry melting profiles of 5S rRNAs from lupin seeds (L.s.) and wheat germ (W.g.) with their structure. It is suggested that the observed differences in thermal unfolding are due to differences in RNA nucleotide sequence and as a consequence in higher order structures. Interesting effects induced by magnesium cation, perprotonated and permethylated sperminium tetracations are discussed. It is suggested that the difference in the stabilizing effect of the three cations results from different mode of their interactions with RNA. "Pure" electrostatic interactions expected for permethylated tetracations are rather weak due to the steric hindrance around each positively charged nitrogen atom. Electrostatic interactions of the other two cations are significantly enhanced by coordination bonding for magnesium and by hydrogen bonding for protonated sperminium cation.     

The Influence of Monovalent Cation Size on the Stability of RNA Tertiary Structures

Many RNA tertiary structures are stable in the presence of monovalent ions alone. To evaluate the degree to which ions at or near the surfaces of such RNAs contribute to stability, the salt-dependent stability of a variety of RNA structures was measured with each of the five group I cations. The stability of hairpin secondary structures and a pseudoknot tertiary structure are insensitive to the ion identity, but the tertiary structures of two other RNAs, an adenine riboswitch and a kissing loop complex, become more stable by 2-3 kcal/mol as ion size decreases. This "default" trend is attributed to the ability of smaller ions to approach the RNA surface more closely. The degree of cation accumulation around the kissing loop complex was also inversely proportional to ion radius, perhaps because of the presence of sterically restricted pockets that can be accessed only by smaller ions. An RNA containing the tetraloop-receptor motif shows a strong (up to ∼ 3 kcal/mol) preference for Na⁺ or K⁺ over other group I ions, consistent with the chelation of K⁺ by this motif in some crystal structures. This RNA reverts to the default dependence on ion size when a base forming part of the chelation site is mutated. Lastly, an RNA aptamer for cobinamide, which was originally selected in the presence of high concentrations of LiCl, binds ligand more strongly in the presence of Li⁺ than other monovalent ions.On the basis of these trends in RNA stability with group I ion size, it is argued that two features of RNA tertiary structures may promote strong interactions with ions at or near the RNA surface: negative charge densities that are higher than that in secondary structures, and the occasional presence of chelation sites, which are electronegative pockets that selectively bind ions of an optimum size.     

Different sequences show similar quaternary interaction stabilities in prohead viral RNA self-assembly

Prohead RNA (pRNA) is an essential component of the self-assembling φ29 bacteriophage DNA packaging motor. Different related species of bacteriophage share only 12% similarity in pRNA sequences. The secondary structure for pRNA is conserved, however. In this study, we present evidence for self-assembly in different pRNA sequences and new measurements of the energetics for the quaternary interactions in pRNA dimers and trimers. The energetics for self-assembly in different pRNA sequences are similar despite very different sequences in the loop-loop interactions. The architecture surrounding the interlocking loops contributes to the stability of the pRNA quaternary interactions, and sequence variation outside the interlocking loops may counterbalance the changes in the loop sequences. Thus, the evolutionary divergence of pRNA sequences maintains not only conservation of function and secondary structure but also stabilities of quaternary interactions. The self-assembly of pRNA can be fine-tuned with variations in magnesium chloride, sodium chloride, temperature, and concentration. The ability to control pRNA self-assembly holds promise for the development of nanoparticle therapeutic applications for this biological molecule. The pRNA system is well suited for future studies to further understand the energetics of RNA tertiary and quaternary interactions, which can provide insight into larger biological assemblies such as viruses and biomolecular motors.     

Experimental Articles

Volcanic ash (Karymskii Volcano, Kamchatka) stimulated growth of the bacterium Rhodovulum sp. A-20s. The interaction between ash, water, media, and bacteria resulted in changes in the chemical composition of the solutions and ash. The ash-water interaction resulted in release of calcium to the solution, as well as in an increase in the proportions of sodium and calcium among the exchange cations of ash. As a result of the ash-medium interaction, calcium and copper were released to the solution; the exchange sodium cations were substituted by calcium and potassium. As a result of the ash-bacteria interaction, the content of copper in the solution decreased, and the exchange cations of calcium and sodium were actively substituted by potassium and magnesium. An increase in the magnesium content among the exchange cations of ash was especially apparent. The products of bacterial metabolism formed mineral-organic complexes with the ash substrate. The data obtained indicate the biogenic transformation of ash, which may lead to the initial phase of formation of clay minerals from volcanic ashes.     

Observations on the use of a cation exchange resin for the preparation of metal-depleted media for lymphocyte culture

A chelating resin specific for divalent cations (Chelex) was used to prepare metal-depleted media for lymphocyte culture. A batch procedure (resin in pH 7.4 phosphate buffer/specimen, 1:1) removed 70-80% of iron, 77-87% of copper and 88-98% of zinc, calcium and magnesium. At variance with other reports, when a resin/specimen ratio of 1:4 was used, iron chelation decreased to 40%, whereas other cation chelation remained unchanged. Best chelation for iron and calcium was obtained at pH 5-6.4; for copper, zinc and magnesium, at pH 7.4-8.0. During the procedure protein content decreased by 8-10%; arginine and lysine by 80%; asparagine, cystine, tyrosine and phenylalanine by 60%, other amino acids by 35%. These new data suggest that cation-depleted media prepared with Chelex may be used to study the effects of cations on lymphocytes in culture, provided that the most appropriate pH and resin/specimen ratio are selected and adequate amino acid replacement is performed. Results on normal human lymphocytes are reported.     

Conformational plasticity of RNA for target recognition as revealed by the 2.15?? crystal structure of a human IgG–aptamer complex

Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer–hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer–protein complexes. Moreover, the aptamer–hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.     

Magnesium binding to yeast ribosomes

This paper describes a theoretical and experimental analysis of the binding of magnesium ions to yeast, ribosomes. In the theoretical considerations the interactions between charges located on a macroion are included. In the calculations these interactions result in a term, in which both the charge and the radius of the macroion are accounted for. It appears that on dissociation of the ribosomes both the charge and the radius change, but in such a way, that the term, which accounts for the electrostatic interactions, remains constant. As a consequence the dissociation can lie neglected in the analyses of the binding experiments. Our experiments indicate that two binding reactions between ribosomes and magnesium ions occur. The endpoints of these reactions correspond to about 0.40 and 1.0 equivalent magnesium per ribosomal phosphate, respectively. The pK values are about 3.8 and 2.2, respectively. The experimental results indicate that the effect, of monovalent cations can be explained as a pure ionic strength effect, though the binding of monovalent cations could not be excluded completely.     

Role of electrostatic interactions in cohesion of bacterial biofilms

Significant decreases in the apparent viscosity of a bacterial biofilm suspension were measured following addition of sodium, potassium, magnesium, or calcium salts, whereas iron salts increased the viscosity. Electrostatic interactions contribute to biofilm cohesion and iron cations are potent crosslinkers of the biofilm matrix.     

The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation

The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing--both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.     

A guide to ions and RNA structure

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. "Diffuse ions" accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A "chelated" ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg(2+) on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.     

Contribution of enthalpy to the energetics of complex formation of aromatic ligands with DNA

The energy contributions of electrostatic, van der Waals interactions, hydrogen bonds, and interactions of charge transfer type to the enthalpy of complex formation of the double-stand DNA with the antitumor antibiotics daunomycin, nogalamycin, and novantron, as well as the mutagens ethidium bromide and proflavine have been calculated. According to the calculations, the van der Waals component (except for nogalamycin) is energetically favorable during complex formation of the antibiotics with DNA, and the contributions of H bonds and electrostatic interactions are unfavorable, with the probability of charge transfer in the complexes being low. It has been shown that the relatively low value of the experimental enthalpy of binding is the sum of components greater in absolute value and different in the sign, which is the cause of large errors in estimating the total enthalpy of complex formation of aromatic ligands with DNA.     

A 43Ca-NMR study of Ca(II)-DNA interactions

High-field ⁴³Ca-nmr is applied to characterize the interactions of calcium ions with double-helical DNA. Under the conditions examined, ⁴³Ca lineshapes are always Lorentzian and single spin-lattice relaxation rates are obtained. The measured transverse and longitudinal relaxation rates are, however, not equal, which implies that the relaxation is in the near-extreme narrowing regime. Relative to the transverse relaxation rate, calcium ions near the DNA exchange rapidly with the bulk solution. The ⁴³Ca linewidths, spin-lattice relaxation rates, and chemical shifts observed over the course of a titration of DNA with calcium salt are not well described by simple electrostatic models. Deviations are most pronounced at low ratios of calcium to DNA phosphate. In contrast, at higher Ca/P ratios, the changes observed are well described by an electrostatic model based on the Poisson–Boltzmann equation. These results suggest that there is a small class of site-bound calcium as well as a large background of delocalized calcium electrostatically associated with the DNA. In contrast to previous studies of ²⁵Mg²⁺–DNA interactions, for which significant site-binding effects were also indicated, it appears rather easy to displace bound ⁴³Ca²⁺ by competition with sodium or magnesium cations. Unfortunately, neither these earlier results nor the present work allows a precise quantitation of the extent of site-bound divalent cation.     

Electrostatic contribution to the bending of DNA

A model is derived that accounts for the short-range electrostatic contribution to the bending of DNA molecule in solution and in complexes with proteins in terms of the non-linear Poisson-Boltzmann equation. We defined that the short-range electrostatic interactions depend on the changes of the polyion surface charge density under deformation, while the long-range interactions depend on the bending-induced changes in distances between each two points along the polyion axis. After an appropriate simplification of the Poisson-Boltzmann equation, the short-range term is calculated separately giving the lower limit for the electrostatic contribution to the DNA persistence length. The result is compared with the theoretical approaches developed earlier [M. Fixman, J. Chem. Phys. 76 (1982) 6346; M. Le Bret, J. Chem. Phys. 76 (1982) 6243] and with the experimental data. The conclusion is made that the results of Fixman-Le Bret, which took into account both types of the electrostatic interactions for a uniformly bent polyion, give the upper limit for the electrostatic persistence length at low ionic strength, and the actual behavior of the DNA persistence length lies between two theoretical limits. Only the short-range term is significant at moderate-to-high ionic strength where our results coincide with the predictions of Fixman-Le Bret. The bending of DNA on the protein surface that is accompanied by an asymmetric neutralization of the DNA charge is also analyzed. In this case, the electrostatic bending energy gives a significant favorite contribution to the total bending energy of DNA. Important implications to the mechanisms of DNA-protein interactions, particularly in the nucleosome particle, are discussed.     

Tb3+ and Ca2+ binding to phosphatidylcholine. A study comparing data from optical, NMR, and infrared spectroscopies.

The paramagnetic and luminescent lanthanides are unique probes of cation-phospholipid interactions. Their spectroscopic properties provide the means to characterize and monitor complexes formed with lipids in ways not possible with biochemically more interesting cations, such as Ca2+. In this work, Tb3+-phosphatidylcholine complexes are described using the luminescence properties of Tb3+, the effect of its paramagnetism on the 31P NMR and 13C NMR spectra of the lipid, and changes in the infrared spectrum of the lipid induced by the cation. There are two Tb3+-phosphatidylcholine complexes with very different coordination environments, as evidenced by changes in the optical excitation spectrum of the lanthanide. The NMR experiments indicate that the two complexes differ in the number of phosphate groups directly coordinating Tb3+. Tb3+ binding induces changes in the phosphodiester infrared bands that are most consistent with bidentate chelation of Tb3+ by each phosphate, whereas Ca2+-induced changes are more consistent with monodentate coordination. The significance of this discrepancy is discussed.     

R-loop stability as a function of RNA structure and size.

The sequence-specific formation of R-loops can be assayed using RNAs which overlap a HindIII cleavage site in a 3.5 kb plasmid. Chemical modification of the displaced DNA strand has permitted stabilization of these R-loops and allowed a systematic investigation of the dependence of these triple-stranded structures on the chain length and structure of the input RNA. RNAs as short as 50 nt form stable R-loops if 5-allylamine uridines (Uaa-RNA) are used in place of normal uridines; normal RNAs must be 100 nt long to form R-loops quantitatively. Since acetic anhydride decreases the hybridization efficiency of Uaa-RNAs, the positive charge of the RNAs must diminish the electrostatic repulsion of the three negatively charged phosphodiester backbones. The dependence of R-loop stability on the length of RNA can be stimulated with a random walk model, which also applies to strand migration within Holiday junctions. R-loop hybridization provides a versatile method to generate single-stranded DNA in a sequence-selective manner.     

Manganese-deoxyribonucleic acid binding modes. Nuclear magnetic relaxation dispersion results.

Ion-DNA interactions are discussed and the applied magnetic field strength dependence of water proton spin-lattice relaxation rates is used to study the Mn(II)-DNA interaction both qualitatively and quantitatively. Associations in which the manganese II (Mn(II)) ion is completely immobilized on the DNA are identified as well as a range of associations in which the ion is only partially reorientationally restricted. Quantitative analysis of the strength of the association in which manganese is immobilized is carried out both with and without a counter-ion condensation correction for electrostatic attraction of the mobile ions. From competition experiments with manganese the relative strengths of the interactions of magnesium and calcium with DNA are found to be identical but less than that of manganese with DNA and the affinity of lithium for DNA is found to be slightly higher than that of sodium. The data demonstrate that the reduced mobility of nonsite-bound ions may have a significant effect on DNA-ion binding analyses performed using magnetic resonance and relaxation methods.     

Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron

Although the active site of group I introns is phylogenetically conserved, subclasses of introns have evolved different mechanisms of stabilizing the catalytic core. Large introns contain weakly conserved 'peripheral' domains that buttress the core through predicted interhelical contacts, while smaller introns use loop-helix interactions for stability. In all cases, specific and non-specific magnesium ion binding accompanies folding into the active structure. Whether similar RNA-RNA and RNA-magnesium ion contacts play related functional roles in different introns is not clear, particularly since it can be difficult to distinguish interactions directly involved in catalysis from those important for RNA folding. Using phosphorothioate interference with RNA activity and structure in the small (249 nt) group I intron from Anabaena, we used two independent assays to detect backbone phosphates important for catalysis and those involved in intron folding. Comparison of the interference sites identified in each assay shows that positions affecting catalysis cluster primarily in the conserved core of the intron, consistent with conservation of functionally important phosphates, many of which are magnesium ion binding sites, in diverse group I introns, including those from Azoarcus and Tetrahymena. However, unique sites of folding interference located outside the catalytic core imply that different group I introns, even within the same subclass, use distinct sets of tertiary interactions to stabilize the structure of the catalytic core.