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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 fidelity.A) Representative phosphorimage of ligation reactions using radiolabeled dCHr8 and bridging oligos with each of the four potential templating nucleotide across from the tethered dC. Lane 1, 25 nt DNA size marker. Lanes 2–5, products of 24 h ligation reactions under optimized conditions using the A2Y bridge template strand indicated above the lanes. Lane 6, unreacted input dCHr8 substrate. The percent of dCHr8 converted to full-length ligation product for each reaction is shown above the gel. B) Yields of adenylates (open bars) and ligated products (filled bars) at 0, 2, and 6 h ligation in the presence of different matching and mismatching A2Y bridge templates (indicated below the plots) in separate reactions with each dXHr8 oligo (indicated within each panel).
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pone-0012368-g004: Ligation fidelity.A) Representative phosphorimage of ligation reactions using radiolabeled dCHr8 and bridging oligos with each of the four potential templating nucleotide across from the tethered dC. Lane 1, 25 nt DNA size marker. Lanes 2–5, products of 24 h ligation reactions under optimized conditions using the A2Y bridge template strand indicated above the lanes. Lane 6, unreacted input dCHr8 substrate. The percent of dCHr8 converted to full-length ligation product for each reaction is shown above the gel. B) Yields of adenylates (open bars) and ligated products (filled bars) at 0, 2, and 6 h ligation in the presence of different matching and mismatching A2Y bridge templates (indicated below the plots) in separate reactions with each dXHr8 oligo (indicated within each panel).

Mentions: Ligation fidelity was first examined by comparing yields for radiolabeled dCHr8 annealed to bridging oligonucleotides with matched (A2G) or mismatched (A2A, A2C and A2T) junction nucleotides. The matched combination yielded over 70% ligated product, while the mismatched combinations yielded 0.5 to 0.9%, for a discrimination of approximately 100-fold (Fig. 4A). This analysis was then expanded to include all combinations of junction nucleotides dX/(A2Y), where dX = dC, dT, dA, dG, dI or dNI in the tethered mononucleotide, and Y = dA, dC, dG or dT in the bridging oligonucleotide. The ligation complexes were assembled by annealing the capture oligo with each of the dXHr8 substrates and with all four A2Y bridge oligonucleotides in separate reactions. Tethered dNI mononucleotide was included in the set because it is reported to pair promiscuously when used in PCR primers [25], [26], [27], [28]. Two additional bridging oligonucleotides—HEG-G or T2G—were included that carried a dG in the pairing position with either HEG or a T2 spacer, respectively, across from the HEG tether to determine the effect of spacer composition on the fidelity.


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 fidelity.A) Representative phosphorimage of ligation reactions using radiolabeled dCHr8 and bridging oligos with each of the four potential templating nucleotide across from the tethered dC. Lane 1, 25 nt DNA size marker. Lanes 2–5, products of 24 h ligation reactions under optimized conditions using the A2Y bridge template strand indicated above the lanes. Lane 6, unreacted input dCHr8 substrate. The percent of dCHr8 converted to full-length ligation product for each reaction is shown above the gel. B) Yields of adenylates (open bars) and ligated products (filled bars) at 0, 2, and 6 h ligation in the presence of different matching and mismatching A2Y bridge templates (indicated below the plots) in separate reactions with each dXHr8 oligo (indicated within each panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0012368-g004: Ligation fidelity.A) Representative phosphorimage of ligation reactions using radiolabeled dCHr8 and bridging oligos with each of the four potential templating nucleotide across from the tethered dC. Lane 1, 25 nt DNA size marker. Lanes 2–5, products of 24 h ligation reactions under optimized conditions using the A2Y bridge template strand indicated above the lanes. Lane 6, unreacted input dCHr8 substrate. The percent of dCHr8 converted to full-length ligation product for each reaction is shown above the gel. B) Yields of adenylates (open bars) and ligated products (filled bars) at 0, 2, and 6 h ligation in the presence of different matching and mismatching A2Y bridge templates (indicated below the plots) in separate reactions with each dXHr8 oligo (indicated within each panel).
Mentions: Ligation fidelity was first examined by comparing yields for radiolabeled dCHr8 annealed to bridging oligonucleotides with matched (A2G) or mismatched (A2A, A2C and A2T) junction nucleotides. The matched combination yielded over 70% ligated product, while the mismatched combinations yielded 0.5 to 0.9%, for a discrimination of approximately 100-fold (Fig. 4A). This analysis was then expanded to include all combinations of junction nucleotides dX/(A2Y), where dX = dC, dT, dA, dG, dI or dNI in the tethered mononucleotide, and Y = dA, dC, dG or dT in the bridging oligonucleotide. The ligation complexes were assembled by annealing the capture oligo with each of the dXHr8 substrates and with all four A2Y bridge oligonucleotides in separate reactions. Tethered dNI mononucleotide was included in the set because it is reported to pair promiscuously when used in PCR primers [25], [26], [27], [28]. Two additional bridging oligonucleotides—HEG-G or T2G—were included that carried a dG in the pairing position with either HEG or a T2 spacer, respectively, across from the HEG tether to determine the effect of spacer composition on the fidelity.

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