Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various genomic tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to insert 5' phosphorylated blunt-end double-stranded DNA to DNA breaks at 3'-recessive ends, gaps, or nicks to form a Y-shaped 3'-branch structure. Therefore, this base pairing-independent ligation is termed 3'-branch ligation (3'BL). In an extensive study of optimal ligation conditions, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency to more than 80%. Ligation efficiency was slightly varied between different donor and acceptor sequences. More interestingly, we discovered that T4 DNA ligase efficiently ligated DNA to the 3'-recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, suggesting a ternary complex formation preference of T4 DNA ligase. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important genomic applications, such as 3'-end labelling by adding a universal sequence; directional tagmentation for NGS library construction that achieve theoretical 100% template usage; and targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

中文翻译：

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3'分支连接：一种将非互补DNA连接到DNA或RNA的凹入或内部3'OH末端的新方法。

核酸连接酶是在合成，修复和重组过程中修复DNA或RNA断裂的关键酶。利用DNA / RNA连接酶的多种活性，已经开发出各种基因组工具。在本文中，我们证明了T4 DNA连接酶具有非常规能力，可将5'磷酸化的平端双链DNA插入3'隐性末端，缺口或缺口的DNA断裂处，形成Y形3'分支结构。因此，这种与碱基配对无关的连接被称为3'分支连接（3'BL）。在最佳连接条件的广泛研究中，连接缓冲液中10％PEG-8000的存在将连接效率显着提高到80％以上。连接效率在不同的供体和受体序列之间略有不同。更有趣的是 我们发现，在DNA / RNA杂合体中，T4 DNA连接酶有效地将DNA连接到RNA的3'末端，而不是DNA的末端，表明T4 DNA连接酶的三元复合物形成偏好。T4 DNA连接酶的这些新特性可以在许多重要的基因组应用中用作广泛的分子技术，例如通过添加通用序列进行3'末端标记；用于NGS库构建的定向标记，可实现理论上100％的模板使用率；以及针对性的RNA NGS文库，具有缓解的基于结构的偏倚和衔接子二聚体问题。例如通过添加通用序列进行3'末端标记；用于NGS库构建的定向标记，可实现理论上100％的模板使用率；以及针对性的RNA NGS文库，具有缓解的基于结构的偏倚和衔接子二聚体问题。例如通过添加通用序列进行3'末端标记；用于NGS库构建的定向标记，可实现理论上100％的模板使用率；以及针对性的RNA NGS文库，具有缓解的基于结构的偏倚和衔接子二聚体问题。