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Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation.

ABSTRACT: Nicking-sealing RNA ligases play a significant biological role in host defense and cellular repair, and have become an important molecular tool in biomedical engineering. Due to the propensity for RNA to form secondary structures, RNA modifying enzymes with elevated optimum temperatures are highly desired. Current characterized double stranded RNA ligases, such as the bacteriophage T4 RNA ligase 2, while possessing good template dependency, are not active at elevated temperatures. The few characterized RNA ligases from thermophiles exhibit high template independency. We synthesize and characterize here, KOD RNA ligase (KOD1Rnl), a thermostable and template dependent RNA ligase from the archaeon, Thermoccocus Kodakarensis. We disclose that a 13 time reduction in template independent ligation can be achieved with the addition of a single stranded DNase, such as RecJ. We also elucidate the effects of the presence of blood proteins on the activity of KOD1Rnl. Template dependent and thermostable RNA ligases, such as KOD RNA ligase, can be utilized in RNA detection, modification and sequencing.

SUBMITTER: Zhang L

PROVIDER: S-EPMC5270536 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation.

Zhang Lei L Tripathi Anubhav A

RNA biology 20161007 1

Nicking-sealing RNA ligases play a significant biological role in host defense and cellular repair, and have become an important molecular tool in biomedical engineering. Due to the propensity for RNA to form secondary structures, RNA modifying enzymes with elevated optimum temperatures are highly desired. Current characterized double stranded RNA ligases, such as the bacteriophage T4 RNA ligase 2, while possessing good template dependency, are not active at elevated temperatures. The few charac ...[more]

PMID: 27715457

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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

Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation.

Publications

Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation.

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