2,6-diaminopurine (Z), a naturally occurring noncanonical nucleotide base found in bacteriophages, enhances DNA hybridization by forming three hydrogen bonds with thymine (T). These distinct biochemical characteristics make it particularly valuable in applications that rely on the thermodynamics of DNA hybridization. However, the practical use of Z-containing oligos is limited by their high production cost and the challenges associated with their synthesis. Here, we developed an efficient and cost-effective approach to synthesize Z-containing oligos of high quality based on an isothermal strand displacement reaction. These newly synthesized Z-oligos are then employed as toehold-blockers in an isothermal genotyping assay designed to detect rare single nucleotide variations (SNV). When compared with their counterparts containing the standard adenine (A) base, the Z-containing blockers significantly enhance the accuracy of identifying SNV. Overall, our innovative methodology in the synthesis of Z-containing oligos, which can also be used to incorporate other unconventional and unnatural bases into oligonucleotides, is anticipated to be adopted for diverse applications, including genotyping, biosensing, and gene therapy.

中文翻译：

---

含 2,6-二氨基嘌呤 (Z) 的立足点探针可提高基因分型灵敏度


2,6-二氨基嘌呤 (Z) 是噬菌体中天然存在的非规范核苷酸碱基，通过与胸腺嘧啶 (T) 形成三个氢键来增强 DNA 杂交。这些独特的生化特性使其在依赖 DNA 杂交热力学的应用中特别有价值。然而，含 Z 寡核苷酸的实际应用受到其高生产成本和合成相关挑战的限制。在这里，我们开发了一种基于等温链置换反应的高效且具有成本效益的方法来合成高质量的含 Z 寡核苷酸。然后将这些新合成的 Z-oligo 在等温基因分型测定中用作立足点阻断剂，旨在检测罕见的单核苷酸变异 (SNV)。与含有标准腺嘌呤 (A) 碱基的对应物相比，含有 Z 的阻断剂显着提高了识别 SNV 的准确性。总体而言，我们在合成含 Z 寡核苷酸方面的创新方法，也可用于将其他非常规和非天然碱基掺入寡核苷酸中，预计将用于多种应用，包括基因分型、生物传感和基因治疗。