Engineered allosteric ribozymes as biosensor components

RNA and DNA molecules can be engineered to function as molecular switches that trigger catalytic events when a specific target molecule becomes bound. Recent studies on the underlying biochemical properties of these constructs indicate that a significant untapped potential exists for the practical application of allosteric nucleic acids. Engineered molecular switches can be used to report the presence of specific analytes in complex mixtures, making possible the creation of new types of biosensor devices and genetic control elements.     

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions.

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.     

Subsite specificity of divalent metal ions to glucosyltransferase

Glucosyltransferase from oral bacteria Streptococcus mutans is the most significant virulent factor in causing dental caries. The enzyme has two subsites. The binding specificity of divalent metal ions to glucosyl or fructosyl subsite was examined using multiple inhibition kinetics. The interaction factor "alpha" identifies whether the two subsites are exclusive or non-exclusive.     

Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3′-terminus. The variants show high cleavage activity in the presence of Mg²⁺, Ca²⁺ and Mn²⁺, lower with Co²⁺ and Sr²⁺ and some variants are also active with Cd²⁺ and Zn²⁺ ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg²⁺ is partially inhibited in the presence of Na⁺, spermidine and some other divalent metal ions. The inert Co(NH₃)₆³⁺ complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.     

Diverse roles of metal ions in acyl-transferase ribozymes

The dependence on metal ions for catalysis is one of the hallmark characteristics of ribozymes. Yet despite this universal reliance, the functional role of divalent ions in promoting RNA catalysis is manifold. In this study we elucidate some different roles metal ions play as catalytic cofactors, by comparing two functionally co-evolved acyl-transferase ribozymes. Earlier studies performed on the in vitro selected acyl-transferase ribozyme, E18 [Suga, H., Cowan, J. A., and Szostak, J. W. (1998) Biochemistry 28, 10118-10125], revealed the requirement of a fully hydrated (outer-sphere) Mg2+ ion for catalytic activity. Interestingly, one class of acyl-transferase ribozymes isolated from the same RNA pool as E18 displays a unique metal dependency and is believed to be interacting with inner-sphere coordinated Mg2+ ions. New results show that one of these inner-sphere coordinating ribozymes, HS01, assumes a cloverleaf secondary structure closely resembling E18, yet apparently facilitates a distinct catalytic mechanism. Furthermore, the nature of the RNA-metal interaction(s) in HS01 seems to be dictating a unique reaction mechanism that exhibits a titratable moiety at a near-neutral pK(a). In light of the critical role metal ions play in biochemistry and the proper function of RNAs, these results compare two distinct manners by which metals serve to promote the catalysis of the same reaction.     

Interaction of DNA with divalent metal ions

The interaction between the native DNA macromolecules and Ca2+, Mn2+, Cu2+ ions in solutions of low ionic strength (10(-3) M Na+) is studied using the methods of differential UV spectroscopy and CD spectroscopy. It is shown that the transition metal ions Mn2+ exercise binding to the nitrogen bases of DNA at concentrations approximately 5 x 10(-6) M and form chelates with guanine of N7-Me(2+)-O6 type. Only at high concentrations in solution (5 x 10(-3) M) do Ca2+ ions interact with the nitrogen bases of native DNA. In the process of binding to Ca2+ and Mn2+ the DNA conformation experiences some changes under which the secondary structure of the biopolymer is within the B-form family. The DNA transition to the new conformation is revealed by its binding to Cu2+ ions.     

Simultaneous stochastic sensing of divalent metal ions

Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.     

Changes in microorganism sensitivity to antibiotics as affected by divalent metal ions

Ca+ and Mg+ in nutrient media significantly influence the results of antibiotic sensitivity determination in microorganisms. The data of the study are indicative of a necessity for the media standardization with respect to the content of bivalent metal ions. It is recommended that agar-agar manufactured by the South Sea Factory (tafuinsky) be used for preparation of nutrient media for determination of microbial sensitivity to antibiotics and Hottinger meat pancreatic digest as the nitrogen source providing the content of 120 mg per cent of amine nitrogen in the medium.     

Interaction of divalent metal ions with human serum albumin

ESR study was carried out of the interaction between Zn2+, Cu2+, Ca2+, Mg2+ ions and human serum albumin (HSA) in the presence of Mn2+ ions which depends on pH. Competitive binding of these ions with "manganese-binding" sites of albumin was shown to depend on pH. An analysis of concentration dependence of binding these ions with human serum albumin confirmed earlier supposition about the nature of the binding sites of Mn2+ ions with HSA.     

Coordinated cations in dipicolinato complexes of divalent metal ions

Several salts of alkali, alkaline earth metal and organic ammonium cations of a complex anion [ML₂]²⁻ {Where L = dipicolinato dianion, M = copper(II), nickel(II) and zinc(II)} are prepared. The coordination effect of [ML₂]²⁻ with the cations such as sodium, potassium, calcium, magnesium, and organic cations namely diammonium cation of 1,5-pentanediamine, diammonium cation of 1,8-octyldiamine, mono ammonium cation of 4-aminobenzylamine are studied by determining their X-ray crystal structures. Depending on the nature of cations, four different types of structures are obtained. When calcium is the cation a polymeric structure with calcium ions bridging the [ML₂]²⁻ is observed. The salts having sodium and potassium cations form polymeric chain like structures by oxo and aqua bridges. In the case of magnesium, the hydrated form of magnesium cations coordinates to [ML₂]²⁻. The organic ammonium salts of [ML₂]²⁻ have the structural features of conventional ionic complexes. These salts easily exchange cations. The organic ammonium salts of [ML₂]²⁻ decomposes to give the corresponding metal oxides at relatively low temperature range 300-450 °C.     

Roles of divalent metal ions in flap endonuclease-substrate interactions

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.     

Interaction of divalent metal ions with four-stranded polyriboinosinic acid

Interaction of Mg2+, Ca2+, Cu2+ ions with the four-stranded poly(I) was studied using differential UV and visible spectroscopies. It was shown that, up to concentrations of approximately 0.1 M, Mg2+ and Ca2+ ions do not bind to heteroatoms of hypoxanthine of the four-stranded poly(I). Cu2+ ions interact with N7 (and/or N1) and O6 (through the water molecule of the hydrate shell of the ion). The latter seems to induce the enolization of hypoxanthine the deprotonation of N1, and, as a result, the transition of the four-stranded helix to single-stranded coils. Single-stranded chains form compact particles with an effective radius of about 100 A.     

Atomic-resolution crystal structures of B-DNA reveal specific influences of divalent metal ions on conformation and packing.

Crystal structures of B-form DNA have provided insights into the global and local conformational properties of the double helix, the solvent environment, drug binding and DNA packing. For example, structures of the duplex with sequence CGCGAATTCGCG, the Dickerson-Drew dodecamer (DDD), established a unique geometry of the central A-tract and a hydration spine in the minor groove. However, our knowledge of the various interaction modes between metal ions and DNA is very limited and almost no information exists concerning the origins of the different effects on DNA conformation and packing exerted by individual metal ions.Crystallization of the DDD duplex in the presence of Mg(2+)and Ca(2+)yields different crystal forms. The structures of the new Ca(2+)-form and isomorphous structures of oligonucleotides with sequences GGCGAATTCGCG and GCGAATTCGCG were determined at a maximum resolution of 1.3 A. These and the 1.1 A structure of the DDD Mg(2+)-form have revealed the most detailed picture yet of the ionic environment of B-DNA. In the Mg(2+)and Ca(2+)-forms, duplexes in the crystal lattice are surrounded by 13 magnesium and 11 calcium ions, respectively.Mg(2+)and Ca(2+)generate different DNA crystal lattices and stabilize different end-to-end overlaps and lateral contacts between duplexes, thus using different strategies for reducing the effective repeat length of the helix to ten base-pairs. Mg(2+)crystals allow the two outermost base-pairs at either end to interact laterally via minor groove H-bonds, turning the 12-mer into an effective 10-mer. Ca(2+)crystals, in contrast, unpair the outermost base-pair at each end, converting the helix into a 10-mer that can stack along its axis. This reduction of a 12-mer into a functional 10-mer is followed no matter what the detailed nature of the 5'-end of the chain: C-G-C-G-A-ellipsis, G-G-C-G-A-ellipsis, or a truncated G-C-G-A-ellipsis Rather than merely mediating close contacts between phosphate groups, ions are at the origin of many well-known features of the DDD duplex structure. A Mg(2+)coordinates in the major groove, contributing to kinking of the duplex at one end. While Ca(2+)resides in the minor groove, coordinating to bases via its hydration shell, two magnesium ions are located at the periphery of the minor groove, bridging phosphate groups from opposite strands and contracting the groove at one border of the A-tract.     

Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes

Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.     

Endothelial cell phagokinesis in response to specific metal ions

Salts of Cu^{I, II}, Ni^II, Sn^II, In^III and a sub-fraction of the cupoprotein ceruloplasmin induced phagokinesis of cultured aortal endothelial cells. A variant aortal endothelial cell line was highly sensitive; cells travelled up to 1000 μm in 24 h in response to 2× 10^?6 M SnCl₂. Other metal ions tested (Zn^II, Co^II, Mn^II, Cr^II, Fe^III, Al^III, Sb^III and Mo^II) were not active. The motility response of endothelial cells to Cu ions in vitro is proposed as a model system for studying early events in neovascularization and as a sensitive assay for detecting angiogenic activity in fractions from cells and tissues.     

Binding of divalent metal ions to calcium-free peroxidase: thermodynamic and kinetic studies

Thermodynamics of binding of divalent metal ions including Ca(2+) , Mg(2+) , Ba(2+) , and Cd(2+) to Ca-free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular-mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca(2+) >Cd(2+) >Mg(2+) >Ba(2+) . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation-time method) for binding of Ca(2+) (as the typical metal ion) to Ca-free HRP, which was found a second-order type having a two-step mechanism involving fast formation of Ca-free HRP/1?Ca(2+) as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M(2+) metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca(2+) ion was occurred on distal and proximal binding sites of Ca-free HRP associated with higher stability energies (E(total) ). Indeed, among the divalent metal ions, Ca(2+) with the highest binding affinity (maximum value of K(bin) and minimum value of ΔG$\rm{{_{bin}^{0}}}$), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.     

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 microM MgCl2. The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80 degrees C in 10 mM MgCl2 and at 70 degrees C in 2 mM MgCl2. A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl2, including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.