Ribozyme Mimics For Catalytic Antisense Strategies

Bashkin, James K.; Xie, Jin; Daniher, Andrew T.; Jenkins, Lisa A.; Yeh, George C.

doi:10.1007/978-94-009-0251-0_24

Cited by 8 publications

(14 citation statements)

References 29 publications

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“…Despite the importance of metal-catalyzed hydrolysis of true DNA and RNA substrates, many mechanistic aspects of these reactions are unresolved. As part of our investigation of hydrolytic RNA cleavage and the design of functional mimics of ribozymes, ,,,,− we report here a detailed kinetic study of the catalytic hydrolysis of adenosine 2‘,3‘-cyclic-monophosphate (cAMP) by Cu(II) terpyridine (Cutrpy). A number of other groups have also reported functional mimics of ribozymes. ,− Here we provide evidence that a single Cutrpy interacts with one cAMP substrate in the rate-determining step of hydrolysis, and that a Cutrpy-bound hydroxide is the reactive catalyst.…”

Section: Introductionmentioning

confidence: 99%

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by Cu^II Terpyridine

et al. 1999

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The hydrolysis and transesterification of RNA are catalyzed by a variety of metal ions, metal complexes, metalloenzymes, and ribozymes. These reactions are of fundamental biochemical importance. However, the role that metal ions play in RNA hydrolysis is not completely understood. We previously showed that aqueous Cu(II) terpyridine (Cutrpy) is effective for both transesterification and hydrolysis of RNA, and we harnessed this reactivity by constructing ribozyme mimics that employ terpyridyl Cu(II) in their active sites. Here we report a detailed kinetic study of the hydrolysis of adenosine-2‘,3‘-cyclic monophosphate (cAMP) by Cutrpy. The reaction is established to be first-order in Cutrpy and first-order in substrate. Catalytic turnover is observed, although product inhibition occurs. Chloride ion also inhibits the reaction, which indicates the disadvantage of using NaCl as an ionic strength buffer in related studies. The pH−rate profile is sigmoidal and implicates the hydroxide form of the catalyst, CutrpyOH+, as the active species. Isotope effects were used to determine whether the metal hydroxide acts as a nucleophile or a base. The solvent deuterium kinetic isotope effect, k H/k D, was measured to be 1.0 ± 0.1 after considering equilibrium isotope effects. To assist the mechanistic interpretation of the measured isotope effects, the fractionation factors for a dianionic phosphorane transition state (methyl phosphate dianion) and hydroxide were evaluated by ab initio quantum chemical calculations. Considering the calculated results along with the relevant experimental fractionation factors, isotope effects were predicted for three overall mechanisms: nucleophilic catalysis, general base catalysis, and specific base/general acid catalysis. We conclude that CutrpyOH+ acts as a nucleophilic catalyst in the hydrolysis of cAMP. This contrasts with the behavior of CutrpyOH+ as a base catalyst in the transesterification of RNA.

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Section: Introductionmentioning

confidence: 99%

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by Cu^II Terpyridine

et al. 1999

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“…Many groups are actively developing RNA cleavage agents with possible medical applications, including the gene-specific, catalytic destruction of viral mRNA. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Generally, catalysts are chosen or rejected based on their ability to cleave dinucleotide substrates, [20][21][22][23] although we and others have reported studies based on metal-promoted cleavage of RNA oligomers and polymers. 9,[24][25][26] The dinucleotide assays may provide misleading information about polynucleotide cleavage, due to the length-dependence of the RNA transesterification reaction (Vide infra).…”

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The Embedded Ribonucleotide Assay: A Chimeric Substrate for Studying Cleavage of RNA by Transesterification

1996

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The cleavage (transesterification) of polyribonucleotides is a process of considerable interest. The use of dinucleotide RNA fragments as substrates for the screening of RNA catalysis agents and mechanistic studies is widespread. This practice may not accurately predict the relative abilities of metal complexes to cleave polyribonucleotide substrates. We report the use of chimeric DNA/RNA molecules, containing RNA nucleotides embedded in DNA sequences, as substrates for studying the transesterification of RNA. The substrates, termed embRNA, display the simplicity of dinucleotide substrates while possessing the multiple phosphate and nucleobase metal-binding sites found in polyribonucleotides. In addition, the DNA residues provide an internal check for oxidative cleavage. The synthesis, purification, and activity of our first-generation embRNA, T11UT7A, is described. T11UT7A is a substrate for the ribonuclease RNase 1, and RNase 1 cleavage provides an excellent measure of the extent of 2‘-deprotection in the synthetic embRNA. Cleavage of T11UT7A by hydroxide and a variety of metal ions and complexes is also reported, and the use of embRNA in kinetic assays is demonstrated. Competitive cleavage of RNA and DNA is built into the embRNA assay. With Pb(II), Ce(III), and Cu(II) reagents, we observed efficient RNA cleavage and no DNA cleavage. Kinetic comparison is made between embRNA T11UT7 and the analogous, all-RNA substrate U19.

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“…The development of artificial nucleases (1)(2)(3)(4)(5)(6)(7)(8) is an area of great interest in chemistry and biology today. Ribozyme mimics (see below) form one class of such biomimetic reagents (9)(10)(11)(12)(13)(14). They employ a DNA (or DNA analog) strand for molecular recognition and an RNA cleavage agent for activity (typically a metal complex that catalyzes the transesterification and/or hydrolysis of RNA).…”

Section: Introductionmentioning

confidence: 99%

Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts

Putnam

2001

Nucleic Acids Research

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Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small molecule catalysis to the design of macromolecular, 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 molecular design principles from small-molecule cleavage to macromolecular cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.

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Ribozyme Mimics For Catalytic Antisense Strategies

Cited by 8 publications

References 29 publications

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by Cu^II Terpyridine

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by Cu^II Terpyridine

The Embedded Ribonucleotide Assay: A Chimeric Substrate for Studying Cleavage of RNA by Transesterification

Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts

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Ribozyme Mimics For Catalytic Antisense Strategies

Cited by 8 publications

References 29 publications

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by CuII Terpyridine

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by CuII Terpyridine

The Embedded Ribonucleotide Assay: A Chimeric Substrate for Studying Cleavage of RNA by Transesterification

Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts

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Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by Cu^II Terpyridine

Catalytic Hydrolysis of Adenosine 2‘,3‘-Cyclic Monophosphate by Cu^II Terpyridine