The enzymatic synthesis of xenobiotic nucleic acids (XNA), which are artificially sugar-modified nucleic acids, is essential for the preparation of XNA libraries. XNA libraries are used in the in vitro selection of XNA aptamers and enzymes (XNAzymes). Efficient enzymatic synthesis of various XNAs can enable the screening of high-quality XNA aptamers and XNAzymes by expanding the diversity of XNA libraries and adding a variety of properties to XNA aptamers and XNAzymes. However, XNAs that form unstable duplexes with DNA, such as arabino nucleic acid (ANA), may dissociate during enzyme synthesis at temperatures suitable for thermophilic polymerases. Thus, such XNAs are not efficiently synthesised by the thermophilic polymerase mutants at the end of the sequence. This undesirable bias reduces the possibility of generating high-quality XNA aptamers and XNAzymes. Here, we demonstrate that polyamine-induced DNA/ANA duplex stabilisation promotes ANA synthesis that is catalysed by thermophilic polymerase mutants. Several polyamines, including spermine, spermidine, cadaverine, and putrescine promote ANA synthesis. The negative effect of polyamines on the fidelity of ANA synthesis was negligible. We also showed that polyamines promote the synthesis of other XNAs, including 2′-amino-RNA/2′-fluoro-RNA mixture and 2′-O-methyl-RNA. In addition, we found that polyamine promotes DNA synthesis from the 2′-O-methyl-RNA template. Polyamines, with the use of thermophilic polymerase mutants, may allow further development of XNA aptamers and XNAzymes by promoting the transcription and reverse transcription of XNAs.

中文翻译：

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多胺通过修饰的嗜热聚合酶突变体促进异生核酸合成

异生核酸（XNA）是人工糖修饰的核酸，其酶促合成对于 XNA 文库的制备至关重要。 XNA 文库用于XNA 适体和酶 (XNAzymes) 的体外选择。各种XNA的高效酶法合成可以通过扩大XNA文库的多样性以及为XNA适体和XNAzyme添加多种特性来筛选高质量的XNA适体和XNAzyme。然而，与 DNA 形成不稳定双链体的 XNA，例如阿拉伯核酸 (ANA)，可能在适合嗜热聚合酶的温度下在酶合成过程中解离。因此，此类 XNA 不能由序列末端的嗜热聚合酶突变体有效合成。这种不良偏差降低了生成高质量 XNA 适体和 XNAzyme 的可能性。在这里，我们证明多胺诱导的 DNA/ANA 双链体稳定可促进由嗜热聚合酶突变体催化的 ANA 合成。几种多胺，包括精胺、亚精胺、尸胺和腐胺，可促进 ANA 合成。多胺对 ANA 合成保真度的负面影响可以忽略不计。我们还表明，多胺促进其他 XNA 的合成，包括 2′-氨基-RNA/2′-氟-RNA 混合物和 2′- O-甲基-RNA。此外，我们发现多胺促进 2′- O-甲基-RNA 模板的 DNA 合成。多胺与嗜热聚合酶突变体一起使用，可以通过促进 XNA 的转录和逆转录来进一步开发 XNA 适体和 XNAzyme。