A general and efficient approach for the construction of RNA oligonucleotides containing a 5′-phosphorothiolate linkage

Li, Nan‐Sheng; Frederiksen, John K.; Koo, Selene C.; Lü, Jun; Wilson, Timothy J.; Lilley, David M. J.; Piccirilli, Joseph A.

doi:10.1093/nar/gkq1265

Cited by 19 publications

(47 citation statements)

References 61 publications

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“…This was constructed by the hybridization of four synthetic oligonucleotides, including the radioactively [5′- 32 P]-labelled substrate strand d in which the scissile phosphate was either unmodified (5′-PO) or incorporated a 5′-phosphorothiolate linkage 16, 25, 27 . Complete sequences for each strand are given in the Supporting Information.…”

Section: Resultsmentioning

confidence: 99%

General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

Kath‐Schorr

Wilson

et al. 2012

J. Am. Chem. Soc.

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The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pKa. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pKa consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data.

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Section: Resultsmentioning

confidence: 99%

General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

Kath‐Schorr

Wilson

et al. 2012

J. Am. Chem. Soc.

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“…20,23 However, in contrast to RNA 3′ - PS linkages, RNA 5′ - PS linkages resist cleavage by snake venom phosphodiesterase. 6 …”

Section: Propertiesmentioning

confidence: 99%

Synthesis, Properties, and Applications of Oligonucleotides Containing an RNA Dinucleotide Phosphorothiolate Linkage

2011

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Conspectus RNA represents a prominent class of biomolecules present in all living systems that plays many essential roles in gene expression, regulation, and development. Accordingly, many biological processes depend on the accurate enzymatic processing, modification, and cleavage of RNA. Understanding the catalytic mechanisms of these enzymes therefore represents an important goal in defining living systems at the molecular level. In this context, RNA molecules bearing 3′- or 5′-S-phosphorothiolate linkages comprise what are arguably among the most incisive mechanistic probes available. They have been instrumental in showing that RNA splicing systems are metalloenzymes and in mapping the ligands that reside within RNA active sites. These models have in turn verified the functional relevance of crystal structures. In other cases, phosphorothiolates have offered mechanistic enzymologists an experimental strategy to circumvent the classic problem of kinetic ambiguity and assign precise roles to catalytic groups as general acids or bases. These insights into macromolecular function are enabled by the synthesis of nucleic acids bearing phosphorothiolate linkages and the unique chemical properties they impart. Here, the synthesis, properties, and applications of oligonucleotides and oligodeoxynucleotides containing an RNA dinucleotide phosphorothiolate linkage are reviewed. Phosphorothioate substitutions, in which sulfur replaces one or both non-bridging oxygens within a phosphodiester linkage, are now widely available and are used routinely in numerous biochemical and medicinal applications. In contrast, phosphorothiolate oligonucleotides, in which sulfur replaces a specific 5′ or 3′ bridging oxygen, have presented a more difficult synthetic challenge, requiring chemical alterations to the attached sugar moiety. In this review, we outline the synthetic strategies used to access these RNA analogues and summarize their responses to chemical and enzymatic cleavage agents, as well as mechanistic insights their use has engendered.

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“…These data provide benchmarks for calculations that require p K a values to simulate chemical mechanisms [45]. Additional efforts should look to the use of sulfur probes to assess the importance of leaving-group lability [46], which can shed light on the assignment of general acid catalysts [46,47]. Recent advances in the application of kinetic isotope effects [48] demonstrate the feasibility of examining ribozyme transition-state structures.…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world

Liberman

Wedekind

2011

Current Opinion in Structural Biology

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Genome sequencing has produced thousands of non-protein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base-pKa shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ1 and S-adenosyl-(L)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.

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A general and efficient approach for the construction of RNA oligonucleotides containing a 5′-phosphorothiolate linkage

Cited by 19 publications

References 61 publications

General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

Synthesis, Properties, and Applications of Oligonucleotides Containing an RNA Dinucleotide Phosphorothiolate Linkage

Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world

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