RNA hydrolysis by Cu(II) complexes: Toward synthetic ribonucleases and ribozymes (original) (raw)
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Journal of the American Chemical Society, 1991
As part of a program to develop chem. RNases that cleave RNA by phosphodiester hydrolysis, a systematic study of covalently linked nucleoside-2,2'-bipyridine (bpy) conjugates is described. 2'-Deoxythymidine was attached at both its 3'- and 5'-positions to bpy derivs. by using phosphoramidite chem., yielding after deprotection ammonium 2'-deoxythymidine 3-[4-(4-methyl-2,2'-bipyridin-4-yl)butyl phosphate] salt (I) and triethylammonium 2'-deoxythymidine 5'-[4-[4'-methyl-2,2'-bipyridin-4-yl]butyl phosphate] (II). 2'-Deoxyuridine was attached to a modified bpy via derivatization of the uracil ring at C-5, giving 5-[3-[[2-[[4-(4'-methyl-2,2'-bipyridin-4-yl)-1-oxobutyl]amino]ethyl]amino]-3-oxopropyl]-2'-deoxyuridine (III). These conjugates and the intermediate bpy derivs. were fully characterized by mass spectrometry and 1H-, 13C-, and 31P NMR spectroscopy. The ability of the bpy moieties to bind Cu(II) was demonstrated spectroscopically. The copper(II) complexes of I-III hydrolyze RNA at 37° and neutral pH. The difference in reactivity of I-III provides the basis for optimizing the activity of hydrolytic chem. nucleases.
Ribozyme mimics as catalytic antisense reagents
Applied Biochemistry and Biotechnology, 1995
Viral and fungal infections and some cancers may be described as diseases that are characterized by the expression of certain unwanted proteins. They could be termed induced genetic disorders, with induction provided by mutation or infection. A comprehensive method to inactivate injurious genes based on their nucleic acid sequences has the potential to provide effective antiviral and anticancer agents with greatly reduced side effects. We describe a chemical approach to such gene-specific pharmaceutical agents. Our initial efforts have been to develop new chemical reagents that can carry out catalytic destruction of specific mRNA sequences. We chose hydrolysis as a chemical means of destruction, because hydrolysis is compatible with living cells. Our sequence-specific catalytic RNA hydrolysis reagents may be described as functional ribozyme mimics. Reactivity is provided by small-molecule catalysts, such as metal complexes. Specificity is provided by oligonucleotide probes. Here we report initial results on the sequence-specific, hydrolytic cleavage of mRNA from the HIV gag gene, using a ribozyme mimic. The reagent is composed of a terpyridylCu(II) complex for cleavage activity and an oligonucleotide for sequence specificity. Index Entries: Hydrolytic cleavage of RNA, sequence-directed, by ribozyme mimic, copper(II) terpyridine-catalyzed; controlling gene expression, gene-specific drugs for, antisense method of, chemoselective reagents for.
Nucleic Acids Research, 2001
Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small mol. catalysis to the design of macromol., biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phenanthroline- and terpyridine-derived reagents. The suppression of hydroxide-bridged dimers and the greater activation of coordinated water by Cu(II) neocuproine (compared with the o-phenanthroline and terpyridine complexes) better allow Cu(II) to reach its catalytic potential as a biomimetic RNA cleavage agent. This work demonstrates the direct mapping of mol. design principles from small-mol. cleavage to macromol. cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.
Further Probing of Cu2+-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes
Molecules, 2019
Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu2+-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu2+-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the...
Molecules, 2021
RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RN...
Beilstein Journal of Organic Chemistry, 2015
Tris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific RNA cleavers. Their mechanism of action is independent of metal ions. Here we investigate conjugates with peptide nucleic acids (PNA). RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates we performed earlier. Although aggregation phenomena are observed in some cases, proper substrate recognition is not compromised. While our previous synthesis of 2-aminobenzimidazoles required an HgO induced cyclization step, a mercury free variant is described herein.
Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme
Biochemistry, 1993
Metal ion requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme have been analyzed. RNA cleavage is observed when Mg2+, Sr2+, or Ca2+ are added to a 40 mM Tris-HC1 buffer, indicating that these divalent cations were capable of supporting the reaction. No reaction was observed when other ions (Mn2+, Co2+, Cd2+, Ni2+, Ba2+, Na+, K+, Li+, NH4+, Rb+, and Cs+) were tested. In the absence of added metal ions, spermidine can induce a very slow ribozyme-catalyzed cleavage reaction that is not quenched by chelating agents (EDTA and EGTA) that are capable of quenching the metal-dependent reaction. Addition of Mn2+ to a reaction containing 2 mM spermidine increases the rate of the catalytic step by at least 100-fold. Spermidine also reduces the magnesium requirement for the reaction and strongly stimulates activity at limiting Mg2+ concentrations. There are no special ionic requirements for formation of the initial ribozyme-substrate complex-analysis of complex formation using native gels and kinetic assays shows that the ribozyme can bind substrate in 40 mM Tris-HC1 buffer. Complex formation is inhibited by both Mn2+ and Co2+. Ionic requirements for the ribozyme-catalyzed ligation reaction are very similar to those for the cleavage reaction. We propose a model for catalysis by the hairpin ribozyme that is consistent with these findings. Formation of an initial ribozyme-substrate complex occurs without the obligatory involvement of divalent cations. Ions (e.g., Mg2+) can then bind to form a catalytically proficient complex, which reacts and dissociates. Because spermidine acts to reduce the Mg2+ requirement but cannot itself promote an efficient reaction, we propose that two different cation-binding sites exist in the active ribozyme-substrate complex. Catalytic RNA molecules, or ribozymes, act to cleave and/ or form phosphodiester linkages in nucleic acid substrates using transesterification or hydrolytic mechanisms. Like protein enzymes that act on nucleic acids, ribozymes show strong requirements for divalent metal ions as cofactors in these reactions (Symons, 1989). For both classes of biocatalyst, this requirement is typically fulfilled by Mg2+. There is strong evidence that divalent metal ions serve as essential components of the catalytic site in reactions catalyzed by both protein enzymes (Fersht, 1985) and ribozymes (Grosshans & Cech, 1989; Dahm & Uhlenbeck, 1991; Kazakov & Altman, 1991; Smith et al., 1992). In addition, ribozymes require cations to function in neutralizing the highly negative charge of the phosphate-sugar backbone to promote ribozyme folding and substrate binding in vitro (Jack et al., 1977; Celander & Cech, 1991). In vivo, this charge-shielding function can sometimes be provided by proteins that facilitate many ribozyme-catalyzed reactions (Reich et al., 1988; Gampel et al., 1989). Ribozymes characterized to date show widely varying selectivity in their utilization of different cations for structural and catalytic purposes. For reactions catalyzed by the Tetrahymena group I ribozyme, a large number of divalent metal ions can satisfy the structural ion requirement (Mg2+, Mn2+, Ca2+, Sr2+, and Ba2+), while only Mg2+ and Mn2+ ions can fulfill thecatalytic requirement (Grosshans 8c Cech, 1989). In a similar manner, the catalytic RNA subunit of RNase P can utilize a wide variety of monovalent cations to stabilize the structure required for complex formation but, as for the This work was supported by grants from the NIH. A.B.-H. was supported by a NATO postdoctoral fellowship.
Nucleic Acids Research, 1993
RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles In the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was Investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAASP transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43 C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives.
Building Blocks for Ribozyme Mimics: Conjugates of Terpyridine and Bipyridine with Nucleosides
The Journal of Organic Chemistry, 1996
The incorporation of the 2,2′:6′,2′′-terpyridyl (terpy) complex of Cu(II) into a deoxyoligonucleotide has led to a functional mimic of ribozymes. The resulting mimic can cleave target RNA in a sequence-directed manner by a hydrolytic mechanism. Here we describe the synthesis and characterization of four modified nucleoside phosphoramidite reagents (7, 10, 14, and 18) that contain pendant 2,2′-bipyridine or terpy ligands. The ligands are attached either to the nucleobase (7, 10) or to the sugar (14, 18). Nucleobase modification was carried out at the C-5 position of 2′-deoxyuridine (dU). One route to sugar modification was performed by synthesis of 18, a 1′functionalized analog of dU based on 1-[3-deoxy-D-psicofuranosyl]uracil. Another route to sugar functionaliztion resulted in 14, a 2′-O-alkyl derivative of adenosine. These modified nucleosides are building blocks for ribozyme mimics. They are designed to deliver hydrolytically active metal complexes across either the major groove (7, 10) or the minor groove (14, 18) of an RNA/DNA duplex.