Kimoto Michiko, Soh Si Hui Gabriella, Hirao Ichiro
Institute of Bioengineering and Nanotechnology, A*STAR, 31 Biopolis Way, The Nanos #07-01, Singapore, 138669, Singapore.
Raffles Institution, 1 Raffles Institution Lane, Singapore, 575954, Singapore.
Chembiochem. 2020 Aug 17;21(16):2287-2296. doi: 10.1002/cbic.202000057. Epub 2020 Apr 22.
Genetic alphabet expansion technology, creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four-letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing) for DNAs containing our UBP, Ds-Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds-Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds-Px pairs in various sequence contexts. This method is useful for UBP applications such as high-affinity DNA aptamer generation and semisynthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, thus suggesting further development of UBPs.
遗传字母表扩展技术，即利用非天然碱基对（UBP）创造新的可复制且具有功能的DNA分子，是合成生物学中一个充满前景的新研究领域。最近，这项技术发展迅速，因此需要一种针对含有UBP的DNA分子的测序方法。然而，所有传统测序方法，如桑格测序法，都是针对四字母DNA分子的。在此，我们提出了一种改进的桑格测序方法（桑格缺口测序），用于对含有我们的UBP（Ds-Px）的DNA进行测序，该UBP在测序峰图中表现为缺口。通过改进测序反应以实现高效的Ds-Px配对，并使用修饰的Px底物，我们开发了一种测序方法，该方法在各种序列背景下对多个Ds-Px对具有更高的持续合成能力和清晰的缺口模式。这种方法对于UBP的应用很有用，例如生成高亲和力DNA适配体以及创建涉及UBP的半合成生物体。此外，通过这项研究，我们发现非天然碱基的侧链对复制过程中UB对的配对效率有很大影响，从而为进一步开发非天然碱基对提供了思路。
