近日，华南理工大学陈庭坚团队报道了滚动圈扩增与扩展的遗传字母表（ExRCA）产生DNA材料结合额外的构建块增强可编程功能化。2025年8月27日出版的《美国化学会志》发表了这一最新研究成果。

非自然碱基对（UBP）的发展扩展了遗传序列，导致DNA信息容量和功能多样性的极大增加。有效扩增含有UBPs的DNA对于UBPs的广泛应用至关重要。滚环扩增作为DNA扩增的主要方法之一，已广泛应用于从生物技术到DNA纳米技术等诸多领域。

研究组探索并揭示了phi29 DNA聚合酶复制具有代表性的UBP， dNaM-dTPT3及其类似物的良好效率和保真度。在此基础上，还建立了扩展遗传字母表滚动圆扩增方法（ExRCA），并提出了几种制备ExRCA循环模板的策略。接下来，研究组演示了ExRCA在含有非天然碱基或UBP的单链和双链DNA的大规模生产或扩增中的应用，以及单链DNA产品用于构建特定位点标记的DNA四面体的主题。

ExRCA在生物技术和DNA材料科学方面的巨大应用潜力进一步得到了证明，研究组利用ExRCA开发了一种超灵敏的生物分子检测方法，可编程的多价适体探针，微调的DNA级联催化剂，以及具有位点特异性交联的功能DNA水凝胶。这项工作不仅为含有非自然核碱基的DNA的等温扩增提供了一种有效的方法，而且还展示了UBP在构建具有增加构建块数量和可编程功能的DNA材料方面具有自然复制效率的独特价值。

附：英文原文

Title: Rolling Circle Amplification with an Expanded Genetic Alphabet (ExRCA) Generates DNA Materials Incorporating Additional Building Blocks for Enhanced Programmable Functionalization

Author: Leli Peng, Fangkai Ye, Ruimin Zhang, Yuhui Du, Qiaoling Liu, Rujie Zhang, Binliang Zhang, Tingjian Chen

Issue&Volume: August 27, 2025

Abstract: The development of unnatural base pairs (UBPs) has expanded the genetic alphabet, resulting in a tremendous increase in the information capacity and function diversity of DNA. Efficient amplification of DNA containing UBPs is crucial for the broad use of the UBPs. As one of the major methods for DNA amplification, rolling circle amplification has been extensively employed in many fields ranging from biotechnology to DNA nanotechnology. Herein, we explored and revealed the good efficiency and fidelity of the phi29 DNA polymerase for replicating a representative UBP, dNaM-dTPT3, and its analogues. Based on this, we established the method of rolling circle amplification with an expanded genetic alphabet (ExRCA), together with several strategies for preparing the circular templates for ExRCA. We next demonstrated the application of ExRCA for the mass production or amplification of single- and double-stranded DNAs containing an unnatural base or UBP, as well as the use of the single-stranded DNA product for constructing a site-specifically labeled DNA tetrahedron. The great application potential of ExRCA in biotechnology and materials science of DNA was further demonstrated by utilizing ExRCA to develop an ultrasensitive method for biomolecule detection, DNA hydrogels site-specifically loaded with various cargos, programmable multivalent aptaprobes, a fine-tuned DNA cascade catalyst, and a functional DNA hydrogel with site-specific cross-links. This work not only offers an efficient method for isothermal amplification of DNA containing unnatural nucleobases but also exhibits the unique value of the UBPs with natural-like replication efficiency in constructing DNA materials with increased number of building blocks and programmable functionalities.

DOI: 10.1021/jacs.5c03404

Source: https://pubs.acs.org/doi/abs/10.1021/jacs.5c03404