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Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

Nickens DG, Bardiya N, Patterson JT, Burke DH - PLoS ONE (2010)

Bottom Line: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate.ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme.The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America.

ABSTRACT

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.

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Experimental system for evaluating minimal tethered junctions.A) Kinetic scheme for ligation showing the three group transfer reactions in the ligation mechanism, as described in the text (bridging strand omitted for clarity). B) Minimal ligation junction including the annealed dCHr8 : capture oligo : AnG bridge oligonucleotides, where n is the number of intervening nucleotides. HEG linker within dCHr8 is shown as an arc. Shaded box, dC∶dG base pair at the ligation junction. Analogous junctions using HEG-G, AnY or TnY bridges are described in the text. C) The dCHr8 oligoribonucleotide used as the downstream oligo of the ligation complexes. Other downstream oligoribonucleotides, referred to collectively as dXHr8, are identical in structure except for the attached nucleobase. D) Identification of ligated product. Ligation product and control unligated dCHr8 were gel purified and digested with T1 ribonuclease at increasing concentrations (wedge above lanes) using 0, 0.01, 0.1, and 1.0 U/µL enzyme. Tethered mononucleotide is radiolabeled (asterisk). Compositions of the major products are shown to the left. Capture oligo strand is represented as a filled rectangle and HEG as an arc; RNA sequences are shown explicitly.
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pone-0012368-g002: Experimental system for evaluating minimal tethered junctions.A) Kinetic scheme for ligation showing the three group transfer reactions in the ligation mechanism, as described in the text (bridging strand omitted for clarity). B) Minimal ligation junction including the annealed dCHr8 : capture oligo : AnG bridge oligonucleotides, where n is the number of intervening nucleotides. HEG linker within dCHr8 is shown as an arc. Shaded box, dC∶dG base pair at the ligation junction. Analogous junctions using HEG-G, AnY or TnY bridges are described in the text. C) The dCHr8 oligoribonucleotide used as the downstream oligo of the ligation complexes. Other downstream oligoribonucleotides, referred to collectively as dXHr8, are identical in structure except for the attached nucleobase. D) Identification of ligated product. Ligation product and control unligated dCHr8 were gel purified and digested with T1 ribonuclease at increasing concentrations (wedge above lanes) using 0, 0.01, 0.1, and 1.0 U/µL enzyme. Tethered mononucleotide is radiolabeled (asterisk). Compositions of the major products are shown to the left. Capture oligo strand is represented as a filled rectangle and HEG as an arc; RNA sequences are shown explicitly.

Mentions: The suitability of a ligation-based approach is governed by the mechanism of ligase enzymes. DNA ligases from viruses, bacteriophage, archaea and eukaryotes couple the strand-joining, or “gap-sealing,” reaction to cleavage of the α−β phosphodiester bond of ATP in a three-step mechanism (Fig. 2A) [19], [20]. In the first step (“enzyme charging”), an active site lysine attacks the α phosphate of ATP, displacing pyrophosphate and forming a metastable phosphoramidate linkage. Pyrophosphate addition can reverse this step, while pyrophosphate hydrolysis makes it effectively irreversible. NAD+ donates the adenylate in the first step of the corresponding bacterial ligases [20]. In the second step (“adenylate formation”), the 5′ phosphate oxyanion from the downstream fragment in the nicked DNA attacks the phosphoramidate α phosphate to regenerate the active site lysine and to produce a 5′,5′-linked adenylate intermediate. In the third step (“gap-sealing”), the 3′ OH of the upstream fragment attacks the 5′ phosphate of the downstream fragment, displacing AMP and sealing the gap. A tethered mononucleotide produced by a kinase ribozyme during selection is expected to be a suboptimal ligation substrate for DNA ligase. Nevertheless, structural contexts other than standard B-form DNA-DNA duplexes are known to be compatible with enzymatic ligation. For example, two RNA fragments annealed to a bridging oligodeoxynucleotide can be ligated using bacteriophage T4 DNA ligase [21]. Mispaired DNA junctions can be ligated under certain conditions [22], as can junctions that contain nucleotide analogs [23].


Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

Nickens DG, Bardiya N, Patterson JT, Burke DH - PLoS ONE (2010)

Experimental system for evaluating minimal tethered junctions.A) Kinetic scheme for ligation showing the three group transfer reactions in the ligation mechanism, as described in the text (bridging strand omitted for clarity). B) Minimal ligation junction including the annealed dCHr8 : capture oligo : AnG bridge oligonucleotides, where n is the number of intervening nucleotides. HEG linker within dCHr8 is shown as an arc. Shaded box, dC∶dG base pair at the ligation junction. Analogous junctions using HEG-G, AnY or TnY bridges are described in the text. C) The dCHr8 oligoribonucleotide used as the downstream oligo of the ligation complexes. Other downstream oligoribonucleotides, referred to collectively as dXHr8, are identical in structure except for the attached nucleobase. D) Identification of ligated product. Ligation product and control unligated dCHr8 were gel purified and digested with T1 ribonuclease at increasing concentrations (wedge above lanes) using 0, 0.01, 0.1, and 1.0 U/µL enzyme. Tethered mononucleotide is radiolabeled (asterisk). Compositions of the major products are shown to the left. Capture oligo strand is represented as a filled rectangle and HEG as an arc; RNA sequences are shown explicitly.
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Related In: Results  -  Collection

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pone-0012368-g002: Experimental system for evaluating minimal tethered junctions.A) Kinetic scheme for ligation showing the three group transfer reactions in the ligation mechanism, as described in the text (bridging strand omitted for clarity). B) Minimal ligation junction including the annealed dCHr8 : capture oligo : AnG bridge oligonucleotides, where n is the number of intervening nucleotides. HEG linker within dCHr8 is shown as an arc. Shaded box, dC∶dG base pair at the ligation junction. Analogous junctions using HEG-G, AnY or TnY bridges are described in the text. C) The dCHr8 oligoribonucleotide used as the downstream oligo of the ligation complexes. Other downstream oligoribonucleotides, referred to collectively as dXHr8, are identical in structure except for the attached nucleobase. D) Identification of ligated product. Ligation product and control unligated dCHr8 were gel purified and digested with T1 ribonuclease at increasing concentrations (wedge above lanes) using 0, 0.01, 0.1, and 1.0 U/µL enzyme. Tethered mononucleotide is radiolabeled (asterisk). Compositions of the major products are shown to the left. Capture oligo strand is represented as a filled rectangle and HEG as an arc; RNA sequences are shown explicitly.
Mentions: The suitability of a ligation-based approach is governed by the mechanism of ligase enzymes. DNA ligases from viruses, bacteriophage, archaea and eukaryotes couple the strand-joining, or “gap-sealing,” reaction to cleavage of the α−β phosphodiester bond of ATP in a three-step mechanism (Fig. 2A) [19], [20]. In the first step (“enzyme charging”), an active site lysine attacks the α phosphate of ATP, displacing pyrophosphate and forming a metastable phosphoramidate linkage. Pyrophosphate addition can reverse this step, while pyrophosphate hydrolysis makes it effectively irreversible. NAD+ donates the adenylate in the first step of the corresponding bacterial ligases [20]. In the second step (“adenylate formation”), the 5′ phosphate oxyanion from the downstream fragment in the nicked DNA attacks the phosphoramidate α phosphate to regenerate the active site lysine and to produce a 5′,5′-linked adenylate intermediate. In the third step (“gap-sealing”), the 3′ OH of the upstream fragment attacks the 5′ phosphate of the downstream fragment, displacing AMP and sealing the gap. A tethered mononucleotide produced by a kinase ribozyme during selection is expected to be a suboptimal ligation substrate for DNA ligase. Nevertheless, structural contexts other than standard B-form DNA-DNA duplexes are known to be compatible with enzymatic ligation. For example, two RNA fragments annealed to a bridging oligodeoxynucleotide can be ligated using bacteriophage T4 DNA ligase [21]. Mispaired DNA junctions can be ligated under certain conditions [22], as can junctions that contain nucleotide analogs [23].

Bottom Line: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate.ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme.The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indiana University, Bloomington, Indiana, United States of America.

ABSTRACT

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.

Show MeSH