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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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Ligation kinetics.A) Ligation reactions were performed at 20°C using 3.3 µM 32P-labeled dXHr8, 4.1 µM capture oligo and 4.1 µM of the Watson-Crick-matched DNA bridging template strands. Squares, ligated product; triangles, adenylated intermediate. Identities of the tethered nucleotide (asterisks) and bridges (in parentheses) are indicated. Values of kinetic rate constants k2 (5′ DNA adenylation) and k3 (gap-sealing) are given in the bottom right panel. B) Ligation reactions for tethered “universal” nucleoside, 5-nitroindole (dNI), were performed under the same conditions as in (A) using 32P-labeled dNIHr8 in the presence of the indicated bridging template. Filled symbols, adenylate; open symbols, ligated product.
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pone-0012368-g005: Ligation kinetics.A) Ligation reactions were performed at 20°C using 3.3 µM 32P-labeled dXHr8, 4.1 µM capture oligo and 4.1 µM of the Watson-Crick-matched DNA bridging template strands. Squares, ligated product; triangles, adenylated intermediate. Identities of the tethered nucleotide (asterisks) and bridges (in parentheses) are indicated. Values of kinetic rate constants k2 (5′ DNA adenylation) and k3 (gap-sealing) are given in the bottom right panel. B) Ligation reactions for tethered “universal” nucleoside, 5-nitroindole (dNI), were performed under the same conditions as in (A) using 32P-labeled dNIHr8 in the presence of the indicated bridging template. Filled symbols, adenylate; open symbols, ligated product.

Mentions: To dissect quantitatively the effects of tethered mononucleotide identity on the rates of adenylate formation and gap-sealing, ligation complexes were assembled with each radiolabeled dXHr8 substrate using the corresponding Watson-Crick paired A2 bridges, and samples were collected every 15 to 30 minutes for 6 h. Product yields were again greatest for tethered dG (96%) and dC (92%) and lowest for tethered dA (24%) and dT (18%), while tethered dI gave an intermediate yield (66%). Fitting the data to kinetic equations for a two-step sequential reaction revealed that adenylation (k2) is faster than gap-sealing (k3) for each of the ligation substrates (Fig. 5A). With the exception of dTHr8, all k2 values are greater than 1.0 h−1. For the gap-sealing step, reactions with tethered dC (0.80 h−1) and dG (0.62 h−1) were the fastest, and the reactions were the slowest with tethered dA (0.075 h−1) and dT (0.06 h−1). The value of the gap-sealing rate for tethered dI (0.21 h−1) was again intermediate. Reactions involving tethered dNI formed little or no ligated product, irrespective of the bridging oligo used (all k3 values<0.05 h−1, and all ligation yields <10%), although adenylation was rapid for all bridge combinations (k2 ranging from 0.6 h−1 to >10 h−1) (Fig. 5B). Thus, the data for both the Watson-Crick combinations and the tethered dNI establish that the gap-sealing step largely determines the yield of ligated product.


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)

Ligation kinetics.A) Ligation reactions were performed at 20°C using 3.3 µM 32P-labeled dXHr8, 4.1 µM capture oligo and 4.1 µM of the Watson-Crick-matched DNA bridging template strands. Squares, ligated product; triangles, adenylated intermediate. Identities of the tethered nucleotide (asterisks) and bridges (in parentheses) are indicated. Values of kinetic rate constants k2 (5′ DNA adenylation) and k3 (gap-sealing) are given in the bottom right panel. B) Ligation reactions for tethered “universal” nucleoside, 5-nitroindole (dNI), were performed under the same conditions as in (A) using 32P-labeled dNIHr8 in the presence of the indicated bridging template. Filled symbols, adenylate; open symbols, ligated product.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2927549&req=5

pone-0012368-g005: Ligation kinetics.A) Ligation reactions were performed at 20°C using 3.3 µM 32P-labeled dXHr8, 4.1 µM capture oligo and 4.1 µM of the Watson-Crick-matched DNA bridging template strands. Squares, ligated product; triangles, adenylated intermediate. Identities of the tethered nucleotide (asterisks) and bridges (in parentheses) are indicated. Values of kinetic rate constants k2 (5′ DNA adenylation) and k3 (gap-sealing) are given in the bottom right panel. B) Ligation reactions for tethered “universal” nucleoside, 5-nitroindole (dNI), were performed under the same conditions as in (A) using 32P-labeled dNIHr8 in the presence of the indicated bridging template. Filled symbols, adenylate; open symbols, ligated product.
Mentions: To dissect quantitatively the effects of tethered mononucleotide identity on the rates of adenylate formation and gap-sealing, ligation complexes were assembled with each radiolabeled dXHr8 substrate using the corresponding Watson-Crick paired A2 bridges, and samples were collected every 15 to 30 minutes for 6 h. Product yields were again greatest for tethered dG (96%) and dC (92%) and lowest for tethered dA (24%) and dT (18%), while tethered dI gave an intermediate yield (66%). Fitting the data to kinetic equations for a two-step sequential reaction revealed that adenylation (k2) is faster than gap-sealing (k3) for each of the ligation substrates (Fig. 5A). With the exception of dTHr8, all k2 values are greater than 1.0 h−1. For the gap-sealing step, reactions with tethered dC (0.80 h−1) and dG (0.62 h−1) were the fastest, and the reactions were the slowest with tethered dA (0.075 h−1) and dT (0.06 h−1). The value of the gap-sealing rate for tethered dI (0.21 h−1) was again intermediate. Reactions involving tethered dNI formed little or no ligated product, irrespective of the bridging oligo used (all k3 values<0.05 h−1, and all ligation yields <10%), although adenylation was rapid for all bridge combinations (k2 ranging from 0.6 h−1 to >10 h−1) (Fig. 5B). Thus, the data for both the Watson-Crick combinations and the tethered dNI establish that the gap-sealing step largely determines the yield of ligated product.

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