Cyclic oligonucleotides are valuable targets with a broad range of potential applications spanning molecular biology and nanotechnology. Of particular importance is their role as templates in the rolling circle amplification (RCA) reaction. We describe three different chemical cyclisation methods for the preparation of single-stranded cyclic DNA constructs. These chemical cyclisation reactions are cheaper to carry out than the enzymatic reaction, and more amenable to preparative scale purification and characterisation of the cyclic product. They can also be performed under denaturing conditions and are therefore particularly valuable for cyclic DNA templates that contain secondary structures. The resulting single-stranded cyclic DNA constructs contain a single non-canonical backbone linkage at the ligation point (triazole, amide or phosphoramidate). They were compared to unmodified cyclic DNA in rolling circle amplification reactions using ϕ-29 and Bst 2.0 DNA polymerase enzymes. The cyclic templates containing a phosphoramidate linkage were particularly well tolerated by ϕ-29 polymerase, consistently performing as well in RCA as the unmodified DNA controls. Moreover, these phosphoramidate-modified cyclic constructs can be readily produced in oligonucleotide synthesis facilities from commercially available precursors. Phosphoramidate ligation therefore holds promise as a practical, scalable method for the synthesis of fully biocompatible cyclic RCA templates. The triazole-modified cyclic templates generally gave lower and more variable yields of RCA products, a significant proportion of which were double-stranded, while the performances of the templates containing an amide linkage lie in between those of the phosphoramidate- and triazole-containing templates.

中文翻译：

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含有单个三唑、酰胺或氨基磷酸酯主链键的环状单链 DNA 构建体的无酶合成及其用作滚环扩增和纳米花形成的模板†

环状寡核苷酸是有价值的靶标，在分子生物学和纳米技术领域具有广泛的潜在应用。特别重要的是它们在滚环扩增 (RCA) 反应中作为模板的作用。我们描述了三种不同的化学环化方法来制备单链环状 DNA 构建体。这些化学环化反应比酶促反应更便宜，并且更适合环状产物的制备规模纯化和表征。它们还可以在变性条件下进行，因此对于含有二级结构的环状 DNA 模板特别有价值。所得的单链环状 DNA 构建体在连接点（三唑、酰胺或氨基磷酸酯）含有单个非规范主链连接。使用 phi-29 和 Bst 2.0 DNA 聚合酶在滚环扩增反应中将它们与未修饰的环状 DNA 进行比较。含有氨基磷酸酯键的环状模板对 phi-29 聚合酶的耐受性特别好，在 RCA 中的表现始终与未修饰的 DNA 对照一样好。此外，这些氨基磷酸酯修饰的环状构建体可以很容易地在寡核苷酸合成设施中从市售前体产生。因此，氨基磷酸酯连接有望成为合成完全生物相容性环状 RCA 模板的实用、可扩展的方法。三唑修饰的环状模板通常产生较低且变化较大的 RCA 产物，其中很大一部分是双链，而含有酰胺键的模板的性能介于氨基磷酸酯模板和含有三唑的模板之间。