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Selection of a novel class of RNA-RNA interaction motifs based on the ligase ribozyme with defined modular architecture.

ABSTRACT: To develop molecular tools for the detection and control of RNA molecules whose functions rely on their 3D structures, we have devised a selection system to isolate novel RNA motifs that interact with a target RNA structure within a given structural context. In this system, a GAAA tetraloop and its specific receptor motif (11-ntR) from an artificial RNA ligase ribozyme with modular architecture (the DSL ribozyme) were replaced with a target structure and random sequence, respectively. Motifs recognizing the target structure can be identified by in vitro selection based on ribozyme activity. A model selection targeting GAAA-loop successfully identified motifs previously known as GAAA-loop receptors. In addition, a new selection targeting a C-loop motif also generated novel motifs that interact with this structure. Biochemical analysis of one of the C-loop receptor motifs revealed that it could also function as an independent structural unit.

SUBMITTER: Ohuchi SP

PROVIDER: S-EPMC2441787 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Selection of a novel class of RNA-RNA interaction motifs based on the ligase ribozyme with defined modular architecture.

Ohuchi Shoji P SP Ikawa Yoshiya Y Nakamura Yoshikazu Y

Nucleic acids research 20080506 11

To develop molecular tools for the detection and control of RNA molecules whose functions rely on their 3D structures, we have devised a selection system to isolate novel RNA motifs that interact with a target RNA structure within a given structural context. In this system, a GAAA tetraloop and its specific receptor motif (11-ntR) from an artificial RNA ligase ribozyme with modular architecture (the DSL ribozyme) were replaced with a target structure and random sequence, respectively. Motifs rec ...[more]

PMID: 18460545

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Project description:BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10) and T(2)>T(3)>T(4)>T(6) approximately T(1)>T(8)>T(10). Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide. CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.

Dataset Information

Selection of a novel class of RNA-RNA interaction motifs based on the ligase ribozyme with defined modular architecture.

Publications

Selection of a novel class of RNA-RNA interaction motifs based on the ligase ribozyme with defined modular architecture.

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