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Template-directed ligation on repetitive DNA sequences: a chemical method to probe the length of Huntington DNA.

ABSTRACT: Several genomic disorders are caused by an excessive number of DNA triplet repeats. We developed a DNA-templated reaction in which product formation occurs only when the number of repeats exceeds a threshold indicative for the outbreak of Chorea Huntington. The combined use of native chemical PNA ligation and auxiliary DNA probes enabled reactions on templates obtained from human genomic DNA.

SUBMITTER: Kern A

PROVIDER: S-EPMC5494559 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Template-directed ligation on repetitive DNA sequences: a chemical method to probe the length of Huntington DNA.

Kern Anika A Seitz Oliver O

Chemical science 20140916 1

Several genomic disorders are caused by an excessive number of DNA triplet repeats. We developed a DNA-templated reaction in which product formation occurs only when the number of repeats exceeds a threshold indicative for the outbreak of Chorea Huntington. The combined use of native chemical PNA ligation and auxiliary DNA probes enabled reactions on templates obtained from human genomic DNA. ...[more]

PMID: 28706635

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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

Template-directed ligation on repetitive DNA sequences: a chemical method to probe the length of Huntington DNA.

Publications

Template-directed ligation on repetitive DNA sequences: a chemical method to probe the length of Huntington DNA.

Similar Datasets

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