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Template-directed chemical ligation to obtain 3'-3' and 5'-5' phosphodiester DNA linkages.

ABSTRACT: Up to now, the direct ligation of two DNA fragments with opposite directions to obtain 3'-3' or 5'-5' phosphate ester bonds is still challenging. The only way to obtain DNA oligonucleotides containing a 3'-3' or 5'-5' inversion of polarity sites is based on professional DNA chemical synthesis. Herein, we demonstrate a convenient template-directed chemical ligation that enables 3'-3' and 5'-5' linkages of two DNA oligonucleotides. This method is based on the assembly of two oligonucleotides on a template in opposite directions through forming antiparallel and parallel duplexes simultaneously, followed by coupling with N-Cyanoimidazole under mild condition. Moreover, on the basis of DNA oligonucleotides with 5'-5' linkage obtained through our template-directed chemical ligation, we developed a new cDNA display technique for in vitro selection of functional polypeptides.

SUBMITTER: Chen H

PROVIDER: S-EPMC3975322 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Template-directed chemical ligation to obtain 3'-3' and 5'-5' phosphodiester DNA linkages.

Chen Haodong H Du Feng F Chen Gangyi G Streckenbach Frank F Yasmeen Afshan A Zhao Yun Y Tang Zhuo Z

Scientific reports 20140404

Up to now, the direct ligation of two DNA fragments with opposite directions to obtain 3'-3' or 5'-5' phosphate ester bonds is still challenging. The only way to obtain DNA oligonucleotides containing a 3'-3' or 5'-5' inversion of polarity sites is based on professional DNA chemical synthesis. Herein, we demonstrate a convenient template-directed chemical ligation that enables 3'-3' and 5'-5' linkages of two DNA oligonucleotides. This method is based on the assembly of two oligonucleotides on a ...[more]

PMID: 24699719

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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 chemical ligation to obtain 3'-3' and 5'-5' phosphodiester DNA linkages.

Publications

Template-directed chemical ligation to obtain 3'-3' and 5'-5' phosphodiester DNA linkages.

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