Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection
Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection
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DOI：
10.1371/journal.pone.0012368
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发表时间：
2010-08-24
期刊：
影响因子：
3.7
通讯作者：
Burke, Donald H.
中科院分区：
文献类型：
--
作者：
Nickens, David G.;Bardiya, Nirmala;Burke, Donald H.
背景：小分子代谢产物（如游离核苷）的激酶核酶的体外选择将需要区分活性和非活性RNA物种的分割系统。虽然磷酸基转移的核酸催化作用已经很好地建立了寡核苷酸底物的5‘或2’ OH的磷酸化，但可扩散小分子的磷酸化尚未得到证实。方法/主要发现：本研究证明了T4 DNA连接酶捕获具有5'磷酸的单脱氧核苷的RNA链的能力。因此，连接反应模拟了核苷激酶（脱氧）核酶选择的分割步骤。与栓系底物的连接比与有缺口的全双工DNA的连接要慢得多，即使连接连接处的脱氧核苷酸是与栓系底物配对的沃森-克里克碱基。当桥接模板链含有未配对的间隔核苷酸时，连接明显增加，根据趋势：A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A（10）和T-2>T-3>T-4>T-6近似于T-1>T-8>T-10。桥接T通常比桥接A产生更高的结扎产物收率。当ATP浓度超过33 μ M时，可能由于解离酶的再腺苷化，导致中间产物的腺苷化积累，导致间隙封闭产物的产量下降。在优化的条件下，T4 DNA连接酶有效地（>90%）连接了连接结3'侧的正确配对或T:G摆动配对的底物，同时对大多数错配对底物的区分率约为100倍。栓接dC和dG的结扎速率和产率最高，其次是栓接脱氧肌苷（dl）和dT，栓接dA的反应最慢。在具有多种底物的复杂反应混合物中，连接酶介导的捕获也观察到相同的动力学趋势。“通用”类似物5-硝基吲哚（dNI）作为拴链核苷酸时不支持结扎。结论/意义：我们的研究结果揭示了T4 DNA连接酶的新活性（模板定向连接拴着的单核苷酸），并建立了这种分配方案，适用于选择磷酸化单核苷底物的核酶。
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 approximate to T-1>T-8>T-10. Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 mu M 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 T:G 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 (dl) 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.