RNA by lanthanide ions

Franklin, S.J. (2001) Lanthanide-mediated DNA hydrolysis. Current Opinion in Chemical Biology, 5,201-208. Komiyama, M., Takeda, N., and Shigekawa, H. (1999) Hydrolysis of DNA and RNA by lanthanide ions mechanistic studies leading to new applications. Chemical Communications, 1443-1451. 

M. Komiyama, N. Takeda, H. Shigekawa, Hydrolysis of DNA and RNA by Lanthanide Ions Mechanistic Studies Leading to New Applications , Chem. Commun., 1443(1999)... 

Sequence-Selective Scission of DNA and RNA by Lanthanide Ions and Their Complexes... 

A full kinetic study of the hydrolysis of c-AMP (248) catalysed by Cu(II) terpyridine (Cutrpy) has revealed that the active species under alkaline conditions is the hydroxide form of the catalyst, (CutrpyOH)+. Although the same species acts as a base catalyst in the transesterification of RNA, here it behaves as a nucleophilic catalyst. Mechanistic studies of the hydrolysis of DNA and RNA by lanthanide ions have revealed that the catalytically active species are dinuclear hydroxo clusters. For example, the mechanism of the Ce(IV) catalysis of DNA hydrolysis (acceleration factor of lO -fold) is considered to involve the facilitated formation of a pentacoordinated intermediate (Scheme 45). However, the Ln(III) catalysis of RNA... 

Ohgoribonucleotides are also efficiently hydrolyzed by lanthanide(III) ions. The scission occurs randomly without any specific base-preference, as is the case in the DNA hydrolysis by Ce(IV). When [LuCblo = 10 mM at pH 7.5 and 30 "C, substrate RNAs are almost completely degraded into small fragments within 1 h. The termini of the RNA fragments are either the 1 -or 3 -monophosphates, and the 2, 3 -cycUc monophosphate termini are hardly formed because they are rapidly hydrolyzed to the monophosphates. It is noteworthy that the rate of RNA hydrolysis by lanthanide ions is drastically dependent on the pH of the solntion. With LuCla,... 

Fig. 31. Two-site RNA cutters based on non-covalent strategy for clipping selected fragment from the RNA substrate. The two phosphodiester linkages in front of the two acridines are simultaneously activated and selectively hydrolyzed by lanthanide ions.

Hydrolytic catalysis by metal ions is also important in the hydrolysis of nucleic acids, especially RNA (36). Molecules of RNA that catalyze hydrolytic reactions, termed ribozymes, require divalent metal ions to effect hydrolysis efficiently. Thus, all ribozymes are metalloenzymes (6). There is speculation that ribozymes may have been the first enzymes to evolve (37), so the very first enzymes may have been metalloenzymes Recently, substitution of sulfur for the 3 -oxygen atom in a substrate of the tetrahymena ribozyme has been shown to give a 1000-fold reduction in rate of hydrolysis with Mg2+ but no attenuation of the hydrolysis rate with Mn2+ and Zn2+ (38). Because Mn2+ and Zn2+ have stronger affinities for sulfur than Mg2+ has, this feature provides strong evidence for a true catalytic role of the divalent cation in the hydrolytic mechanism, involving coordination of the metal to the 3 -oxygen atom. Other examples of metal-ion catalyzed hydrolysis of RNA involve lanthanide complexes, which are discussed in this volume. 

Structure Probes.—The introduction of fluorescing labels into nucleic acids can yield valuable structural information, and both organic compounds and metal ions [Tb (ref. 136) and Eu (ref. 137)] have been used as fluorescent probes for tRNA and other polynucleotides. The degree of secondary structure in RNA has been estimated from Raman scattering by the phosphate group vibrations. A number of n.m.r. studies have appeared, but discussion of these is more suited to a review on n.m.r. spectroscopy. Lanthanide ions have been used as contact shift reagents to probe tRNA structure. ... 

An intruiging approach to site-selective RNA cleavage was reported by Kuzuya et al. [130]. In contrast to the previons examples, in this system the metal complex is free in solution (not localized). Site-selective activation for hydrolysis at single positions in a target RNA strand is achieved by covalent attachment of an acridine moiety to the template DNA strand, which canses local perturbation in the hybridized RNA strand opposite this position [131]. RNA strand scission is very site-specific for the localized perturbation (Fig. 9b). This approach was optimized for various metal ions inclnding Ca and Mg % transition metal ions, and lanthanide ions [132, 133]. Two sites in a single RNA conld be activated by incorporation of two acridines in the DNA template [134], and the rate of the process was further enhanced by the attachment of a ligand for Lu in close proximity to the acridine activator [135]. 

As shown in fig. 13, the logarithm of the rate of RNA hydrolysis steeply increases with increasing pH, up to pH 8, and then reaches a plateau. The slope in the steep region is greater than 2. The experimental points fairly fit the theoretical line (the solid line) which shows the equilibrium concentration of [Nd 2(OH)2]". The other species never satisfy the experimental results. Undoubtedly, this bimetallic cluster is the active species for the RNA hydrolysis. In order to hydrolyze RNA efiiciently, the bimetallic structure is essential, as is the case in DNA hydrolysis. The concentration of this active species is strongly dependent on pH, since it is accompanied by the release of two protons. Because of this, the rate of RNA hydrolysis by lanthanide(III) ions is so highly pH dependent. 

The pH-rate constant profiles for the RNA hydrolysis by all the other lanthanide(III) ions have similar shapes, and are composed of (i) a steep straight line at lower pH and (ii) a plateau at the higher pH values (see fig. 14). The straight line corresponds to the formation of the active species [R 2(OH)2] , whereas its formation is completed in the plateau region at higher pH. As the atomic number of lanthanide ion increases, these profiles gradually shift towards the lower pH side. Accordingly, the concentration of the active species at pH 7 increases with... 

Isolated rat liver RNA polymerase is highly inhibited by La (Novello and Stirpe, 1969). Pr (N03)j inhibited RNA synthesis in liver cell nuclei also RNA polymerase and messenger RNA. In isolated ribosomes, protein synthesis fell 60% in 24 hours and 80% in 48 hours but returned to normal in 96 hours. After 24 hours, polyuridylic acid decreased the inhibition from 60 to 20% (Oberdisse et al., 1974). Eu has been used as a fluorescent probe for transfer RNA (Wolfson and Kearns, 1975). The excitation spectra of Eu and Tb are enhanced by E. coli tRNA Yb, Sm and Gd resemble Mg in enhancing 4-thiouridine fluorescence (Kayne and Cohn, 1974). The binding of paramagnetic lanthanide ions (Eu, Pr, Dy) to yeast phenylalanine-specific tRNA causes shifts in some of the resonances in the low-field NMR spectrum of this molecule (Jones and Kearns, 1974). 

The first step in RNA hydrolysis is the intramolecular nucleophilic attack of the phosphorus atom by the 2 -OH of ribose. This step is activated by the coordination of the phosphodiester linkage in RNA to the lanthanide(III) ion in the bimetallic cluster [R 2(OH)2] , since the electrons are withdrawn by the metal ion from the phosphorus atom. This electron withdrawal promotes the electrophilicity of the P atom, although it is not so drastic as the effect achieved by the Ce(IV) in DNA hydrolysis (cf. sect. 5). Furthermore, the hydroxide ion bound to another lanthanide(III) ion in the bimetallic cluster functions as a general base catalyst, and enhances the electrophilicity of the 2 -OH by removing its proton. Alternatively, the 2 -OH is directly coordinated to this metal ion, and its dissociation to alkoxide ion is facilitated. In this way, both the nucleophilic center (the oxygen in the 2 -OH) and the electrophilic center (the phosphorus atom) are simultaneously activated by the bimetallic cluster, and thus the intramolecular nucleophilic attack proceeds efficiently. 

Fig. 26. Site-selective RNA scission by combinations of the type-I activator and lanthanide(III) ion. Lane 1, La(III) only lane 2, DNApi-S/La(III) lane 3, DNApi-Aor/La(III) lane 4, Eu(III) only lane 5, DNAfi-S/Eu(III) lane 6, DNApi-Acr/Eu(III) lane 7, Lu(III) only lane 8, DNAyi-S/La(III) lane 9, DNApi-Aor/Lu(III). At pH 8.0 and 37 °C for 2 h [RNA,]o = 1, [DNAfi-S]o = [DNApi-AcrJo = 10, and [LnCljJo = 100 (jM [NaClJo = 200 mM. R, RNAj only H, alkaline hydrolysis Tj, RNase Tj digestion C, control reaction in buffer solution.

Nucleotides, in many cases as their metal ion complexes, are involved in a great variety of enzymatic reactions either as substrates or as cofactors. In addition they may be viewed as the monomers of DNA and RNA. Lanthanide complexes of nucleotides have been extensively studied by R.J.P. Williams and his coworkers at Oxford. The interest in these complexes is two fold. The nucleotide conformation in solution can be elucidated by NMR from lanthanide induced chemical shifts and line-broadenings, (Barry et al., 1971). Lanthanide-nucleotide complexes may act as competitive inhibitors in enzymatic reactions, (Tanswell et al., 1974), and can be used as paramagnetic probes in the mapping of their binding site on the enzyme, (Tanswell et al., 1976). 

Lanthanide(III) and other elements in lanthanide series are very effective catalysts for the hydrolysis of the phosphodiester linkages in RNA, whereas nonlanthanide metal ions are virtually inactive." The pseudo-first-order rate constant for the hydrolysis of adenylyl-(3 5 )adenosine (ApA) by LuCls (5 mM) at pH 7.2 and 30°C is 0.19 min", which gives 10 -fold rate acceleration compared to rate of hydrolysis of ApA under the same reaction conditions but in the absence of LuClj. The product is an equimolar mixture of adenosine and... 

---