An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis

Background: Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy.Results: Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ionindependent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH.Conclusions: Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metalcation-independent chemical step, such as breaking the 5′ oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.     

Atom‐Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active‐Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes

The pistol RNA motif represents a new class of self‐cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre‐catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53–U54 cleavage site. While the N1 of G40 is within 3.4 Å of the modeled G53 2′‐OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid–base catalysis, the function of A32 is less clear. We present evidence from atom‐specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2′‐OH of A32 seems crucial for the proper positioning of G40 through a H‐bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner‐sphere coordination of the active‐site Mg²⁺ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg²⁺ playing a catalytic role in cleavage.     

Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme

Background: RNA and DNA are polymers that lack the diversity of chemical functionalities that make proteins so suited to biological catalysis. All naturally occurring ribozymes (RNA catalysts) that catalyze the formation, transfer and hydrolysis of phosphodiesters require metal-ion cofactors for their catalytic activity. We wished to investigate whether, and to what extent, DNA molecules could catalyze the cleavage (by either hydrolysis or transesterification) of a ribonucleotide phosphodiester in the absence of divalent or higher-valent metal ions or, indeed, any other cofactors.Results: We performed in vitro selection and amplification experiments on a library of random-sequence DNA that incorporated a single ribonucleotide, a suitable site for cleavage. Following 12 cycles of selection and amplification, a ‘first generation’ of DNA enzymes (DNAzymes) cleaved their internal ribonucleotide phosphodiesters at rates ∼ 10⁷-fold faster than the spontaneous rate of cleavage of the dinucleotide ApA in the absence of divalent cations. Re-selection from a partially randomized DNA pool yielded ‘second generation’ DNAzymes that self-cleaved at rates of ∼ 0.01 min⁻¹ (a 10⁸-fold rate enhancement over the cleavage rate of ApA). The properties of these selected catalysts were different in key respects from those of metal-utilizing ribozymes and DNAzymes. The catalyzed cleavage took place in the presence of different chelators and ribonuclease inhibitors. Trace-metal analysis of the reaction buffer (containing very high purity reagents) by inductively coupled plasma-optical emission spectrophotometry indicated that divalent or higher-valent metal ions do not mediate catalysis by the DNAzymes.Conclusions: Our results indicate that, although ribozymes are sometimes regarded generically to be metalloenzymes, the nucleic acid components of ribozymes may play a substantial role in the overall catalysis. Given that metal cofactors increase the rate of catalysis by ribozymes only ∼ 10²−10³-fold above that of the DNAzyme described in this paper, it is conceivable that substrate positioning, transition-state stabilization or general acid/base catalysis by the nucleic acid components of ribozymes and DNAzymes may contribute significantly to their overall catalytic performance.     

Peptidyl-transferase ribozymes: trans reactions,structural characterization and ribosomal RNA-like features

Background: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an ‘RNA world’ was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids.Results: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular (‘trans’) peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety.Conclusions: An in vitro-selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the ‘helical wheel’ portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.     

Crucial Roles of Two Hydrated Mg2+ Ions in Reaction Catalysis of the Pistol Ribozyme

Pistol ribozymes constitute a new class of small self‐cleaving RNAs. Crystal structures have been solved, providing three‐dimensional snapshots along the reaction coordinate of pistol phosphodiester cleavage, corresponding to the pre‐catalytic state, a vanadate mimic of the transition state, and the product. The results led to the proposed underlying chemical mechanism. Importantly, a hydrated Mg²⁺ ion remains innersphere‐coordinated to N7 of G33 in all three states, and is consistent with its likely role as acid in general acid base catalysis (δ and β catalysis). Strikingly, the new structures shed light on a second hydrated Mg²⁺ ion that approaches the scissile phosphate from its binding site in the pre‐cleavage state to reach out for water‐mediated hydrogen bonding in the cyclophosphate product. The major role of the second Mg²⁺ ion appears to be the stabilization of product conformation. This study delivers a mechanistic understanding of ribozyme‐catalyzed backbone cleavage.     

Isolation of novel ribozymes that ligate AMP-activated RNA substrates

Background: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5′-5′-pyrophosphate ‘capped’ RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5′-monophosphate (AMP) may be a vestige of ‘RNA world’ catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated.Results: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10¹⁵ RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5′-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of ∼ 5 × 10⁵ over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3′-5′-phosphodiester bonds and were highly specific for activation by AMP at the ligation site.Conclusions: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.     

Examples of cation exchange in new ionic oxovanadium(IV) complexes with anions of cyclobutane-1,1-dicarboxylic acid

The results on the synthesis and study of the crystal structures of compounds based on anionic fragments {VO(Cbdc)₂}^2– formed by oxovanadium(IV) (vanadyl, VO²⁺) and two chelate-bound anions of cyclobutane-1,1-dicarboxylic acid (H₂Cbdc = C₄H₆(COOH)₂) are presented. The use of ammonium cation NH₄⁺ as a counterion in the synthesis leads to the formation of the mononuclear complex (NH₄)₂[VO(Сbdc)₂(H₂O)] · 2H₂O (I). In the case of K⁺ cation, compound [K₄(VO)₂(Сbdc)₄(H₂O)₄] _n (II) with the 3D polymeric crystal structure is formed. The reaction of compound II with Mg(NO3)2 · 6H₂O in an aqueous solution involves the partial substitution of K⁺ by Mg²⁺ cations to form 1D polymeric compound {[KMg_0.5(VO)(Сbdc)₂(H₂O)_6.5] · 3H₂O} _n (III), while a similar reaction of compound I does not afford the product of substitution of NH₄⁺ by Mg²⁺ cations (CIF files CCDC 1551021–1551023 for compounds I–III, respectively).     

Rational design of allosteric ribozymes

Background: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes.Results: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created.Conclusions: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation — a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.     

Heats of adsorption of water on vermiculite having large singly-charged cations in the exchange complex

The isotherms and differential heats of adsorption of water vapor on K-, NH₄-, Rb-, and Cs-vermiculites have been studied by means of a Calvet microcalorimeter having a microweighing adsorption attachment. The results are interpreted taking into account that the large cations K₊, NH₄ ⁺, Rb⁺, and especially Cs⁺, may not replace the initial exchange complexes (Na⁺ or Mg²⁺) of the mineral completely, so that besides hydration of the principal exchange cations the reaction of water molecules being adsorbed with the residual Na⁺ or Mg⁺ cations also takes place. The presence of a certain number of the initial cations (Na⁺ or Mg²⁺) in the Cs form of vermiculite is confirmed by the results of studying the ion exchange equilibria on the Na and natural (Mg) forms of the mineral involving the participation of the Cs₊ ions. The nature of the variation in the dependence of the differential heats of adsorption with an increase in the amount of adsorbed substance indicates the segregation (isolation) of the principal (K⁺, NH₄ ⁺, Rb⁺, Cs⁺) and the residual (Na⁺, Mg²⁺) exchange cation in the structure of the mineral.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 22, No. 1, pp. 91–96, January–February, 1986.     

A Mini‐Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class

Nucleolytic ribozymes catalyze site‐specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild‐type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine‐6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pK_a value of 5.1 was determined for a ¹³C2‐labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pK_a of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine‐6 in the catalytic mechanism besides the previously identified invariant guanine‐48 and a Mg²⁺ ion, both of which are directly coordinated to the non‐bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn²⁺ or Cd²⁺ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X‐ray structure of a 2′‐OCH₃‐U5 modified twister ribozyme.     

Use of zwitterionic micelles in the eluent II: a new approach to ion chromatographic analysis of inorganic cations in biological fluids with direct sample injection

A new ion chromatographic (IC) technique has been developed for the determination of inorganic cations in biological fluids with direct sample injection. This involved the use of a mixed zwitterionic-micelle/electrolyte solution as an eluent. The proteins in the sample became bound to the zwitterionic micelles in the eluent and were thus eliminated from the column. The cations were separated by cation exchange. This method is ideal for the on-line, simultaneous determination of common inorganic cations (Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺) in urine and serum samples. Such an application was demonstrated experimentally. Non-suppressed conductivity was used for analyte detection. The detection limits obtained using this IC system were 2.94, 5.22, 34.9, 32.6, and 56.7 μg/L for Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺, respectively.     

Synthesis and Thermal Decomposition Behaviors of Magnesium Borohydride Ammoniates with Controllable Composition as Hydrogen Storage Materials

An ammonia‐redistribution strategy for synthesizing metal borohydride ammoniates with controllable coordination number of NH₃ was proposed, and a series of magnesium borohydride ammoniates were easily synthesized by a mechanochemical reaction between Mg(BH₄)₂ and its hexaammoniate. A strong dependence of the dehydrogenation temperature and purity of the released hydrogen upon heating on the coordination number of NH₃ was elaborated for Mg(BH₄)₂?x NH₃ owing to the change in the molar ratio of H^δ+ and H^δ?, the charge distribution on H^δ+ and H^δ?, and the strength of the coordinate bond N:→Mg²⁺. The monoammoniate of magnesium borohydride (Mg(BH₄)₂?NH₃) was obtained for the first time. It can release 6.5 % pure hydrogen within 50 minutes at 180 °C.     

Water Activities, Osmotic and Activity Coefficients and Some Correlation of Aqueous Mixtures of Alkaline-Earth and Ammonium Nitrates, NH4NO3-Y(NO3)2-H2O with Y ≡ Ba2+, Mg2+ and Ca2+ at T = 25 °C

The thermodynamic properties of the mixed aqueous electrolyte of ammonium and alkaline earth metal nitrates have been studied using the hygrometric method at 25?°C. The water activities of these {yNH₄NO₃+(1?y)Y(NO₃)₂}(aq) systems with Y ≡ Ba²⁺, Mg²⁺ and Ca²⁺ were measured at total molalities ranging from 0.10 mol?kg^?1 to saturation for different NH₄NO₃ ionic-strength fractions of y=0.20, 0.50 and 0.80. These data allow the calculation of osmotic coefficients. From these measurements, the ionic mixing parameters are determined and used to calculate the solute activity coefficients in the mixtures at different ionic-strength fractions. The results of these ternary solution measurements are compared with those for binary solutions of the alkaline earth nitrates of magnesium, calcium and barium with ammonium nitrates. The behavior of the aqueous electrolyte solutions containing mixtures of barium or calcium or magnesium with ammonium nitrates are correlated and show that ionic interactions are more important for the system containing Mg²⁺ than for Ca²⁺ or Ba²⁺. The trends are mainly due to the effects of the ionic size, polarizability and the hydration of the ions in these solutions.     

Bis(ammonium) tris­(hexa­aqua­magnesium) tetra­kis(hydrogen phosphite)

The structure of the title compound, (NH₄)₂[Mg(H₂O)₆]₃(HPO₃)₄, consists of [Mg(H₂O)₆]²⁺ and (NH₄)⁺ cations and (HPO₃)²⁻ anions held together by an intricate network of hydrogen bonds involving all H atoms except for one linked directly to a P atom. The Mg²⁺ cations are octa­hedrally coordinated by six water mol­ecules. One of the Mg atoms is located on a site with 2/m symmetry, whereas the other Mg atom and the P and N atoms occupy sites with m symmetry.     

Luminophore-immobilized mesoporous silica for selective Hg sensing

Novel mesoporous silica-immobilized rhodamine (MSIR) and silica particle-immobilized rhodamine (SPIR) anchored by a tren (N(CH₂CH₂NH₂)₃) were synthesized. The binding and adsorption abilities of both MSIR and SPIR for metal cations were investigated with fluorophotometry and ion chromatography, respectively. Both MSIR and SPIR show selectivity for Hg²⁺ ion over other metal cations because the Hg²⁺ ion selectively induces a ring opening of the rhodamine fluorophores. The sensitivity of the MSIR for Hg²⁺ ion is greater than that of the SPIR and the MSIR adsorbs 70% of Hg²⁺ ion while the SPIR does only 40%. The MSIR can be also easily recovered by treatment of a solution of TBA⁺OH⁻. For the application of Hg²⁺ detection in the environmental field, the MSIR-coated glass plate is also developed and exhibits an excellent function in visual and fluorescence changes with Hg²⁺ ion.     

New coumarin-based sensor molecule for magnesium and calcium ions

A new coumarin-based sensor molecule (L₁) has been synthesized and this was found to bind calcium and magnesium ions more effectively as compared to other alkali/alkaline earth/lanthanide and certain transition metal ions. A significant enhancement in fluorescence intensity was observed on binding to Ca²⁺ and Mg²⁺ ions; while a minor quenching was observed for weakly bound Hg²⁺, Ni²⁺, Fe³⁺, and Co²⁺ ions. PET process, coupled with the ICT process, is proposed to explain the observed spectral response.     

Two new zincophosphates, (H3NCH2CH2NH3)2[Zn(μ-PO4)2] and (NH4)[(H3N)Zn{(μ-PO4)Zn}3]: Crystal structures and relationships to similar open framework zinco- and aluminophospates

Two new zincophosphates, bis(ethylenediammonim) catena-bis(μ-phosphato)zincate, (H₃NCH₂CH₂NH₃)₂[Zn(μ-PO₄)₂] (1), and ammonium ammine-tris(μ-phosphato)tetrazincate, (NH₄)[(H₃N)Zn{(μ-PO₄)Zn}₃] (2), were synthesized under hydrothermal conditions and their crystal structures were determined by single-crystal X-ray diffraction analysis. The crystal structure of 1 consists of infinite macroanionic ZnP₂O₈⁴⁻ chains, running along the [0 0 1] direction, and diprotonated ethylenediammonium cations, H₂en²⁺. The crystal structure of 2 is built up from ZnO₄, Zn(NH₃)O₃ and PO₄ vertex-sharing tetrahedra connected to form an open 3D framework. The ammonium groups, NH₄⁺, are located in the channels formed by 8M-rings extending along [1 0 0]. In order to study vibrational behavior of H₂en²⁺ and NH₄⁺ cations, NH₃ molecules in 1 and 2, single-crystal Raman spectra were obtained. Structural, chemical and topological similarities to the other open framework zinco- and aluminophosphates incorporating different guest species are discussed.     

Effect of magnesium cations on the activity and stability of β-galactosidases

It was shown that the presence of magnesium cations in the reaction mixture increases, approximately twofold, the activity of bacterial Escherichia coli and yeast Kluyveromyces lactis β-galactosidases but does not affect the activity of bovine liver and fungous Penicillium canescens β-galactosidases. The catalytic constants for E. coli and yeast K. lactis β-galactosidases in the presence of 0.01 M and in the absence of Mg²⁺ cations were determined (490 and 220 s^?1 and 59.8 and 37.4 s^?1, respectively). It was shown that the Michaelis constants for these two enzymes are higher in the presence of Mg²⁺ cations, that the thermal stability of E. coli and K. Lactis β-galactosidases is higher in the presence of 0.01 M Mg²⁺, and that the effective rate constants of thermal inactivation of the enzymes are two-to eightfold lower, depending on conditions, in the presence of Mg²⁺ cations. The maximum stabilizing effect of magnesium cations was observed at weak alkaline pH values (7.5–8.5).     

Thermodynamic Properties of Quaternary Aqueous Solutions of Chlorides Charge-type 1-1*1-1*2-1: NH4Cl + NaCl + YCl2 + H2O with Y ≡ Mg2+, Ca2+, and Ba2+

Thermodynamic properties of quaternary aqueous solutions of mixed chlorides of 1-1*1-1*2-1 charge type with the cations (Na⁺, NH₄ ⁺; Mg²⁺, Ca²⁺, Ba²⁺) were determined using the hygrometric method. The quaternary systems NH₄Cl + NaCl + MgCl₂ + H₂O, NH₄Cl + NaCl + CaCl₂+ H₂O, and NH₄Cl + NaCl + BaCl₂ + H₂O have been studied at 25 °C. The water activities were measured at total molalities from 0.44 mol⋅kg⁻¹ to saturation for different ionic-strength fractions y of NH₄Cl, y=(0.20,0.50,0.80), and different ionic strength ratios z for other solutes, z=(0.20,0.50 and 0.80) for each value of y. The obtained data allows the calculation of osmotic coefficients.     

Preparation of a hybrid monolithic stationary phase with allylsulfonate for the rapid and simultaneous separation of cations in capillary ion chromatography

A hybrid monolithic column with sulfonate functionality was successfully prepared for the simultaneous separation of common inorganic cations in ion‐exchange chromatographic mode through a simple and easy single‐step preparation method. The strong cation‐exchange moieties were provided directly from allylsulfonate, which worked as an organic monomer in the single‐step reaction. Inorganic cations (Li⁺, Na⁺, K⁺, NH₄⁺, Cs⁺, Rb⁺, Mg²⁺, Ca²⁺, and Sr²⁺) were separated satisfactorily by using CuSO₄ as the eluent with indirect UV detection. The allysulfonate hybrid monolith showed a better performance in terms of speed and pressure drop than the capillary packed column. The number of theoretical plates achieved was 19 017 plates/m (in the case of NH₄⁺ as the analyte). The relative standard deviations (n = 6) of both retention time and peak height were less than 1.96% for all the analyte cations. The allysulfonate hybrid monolithic column was successfully applied for the rapid and simultaneous separation of inorganic cations in groundwater and the effluent of onsite domestic wastewater treatment system.