Zhang Qiao, Xia Kai, Jiang Meng, Li Qingting, Chen Weigang, Han Mingzhe, Li Wei, Ke Rongqin, Wang Fei, Zhao Yongxing, Liu Yuehua, Fan Chunhai, Gu Hongzhou
Fudan University Shanghai Cancer Center, and the Shanghai Key Laboratory of Medical Epigenetics, Institutes of Biomedical Sciences, Shanghai Stomatological Hospital, Fudan University, Shanghai, 200433, China.
Department of Chemical Biology, School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules, National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai, 201108, China.
Angew Chem Int Ed Engl. 2023 Jan 26;62(5):e202212011. doi: 10.1002/anie.202212011. Epub 2022 Dec 22.
Synthetic single-stranded (ss) DNA is a cornerstone for life and materials science, yet the purity, quantity, length, and customizability of synthetic DNA are still limiting in various applications. Here, we present PECAN, paired-end cutting assisted by DNAzymes (DNA enzymes or deoxyribozymes), which enables mass production of ssDNA of arbitrary sequence (up to 7000 nucleotides, or nt) with single-base precision. At the core of PECAN technique are two newly identified classes of DNAzymes, each robustly self-hydrolyzing with minimal sequence requirement up- or down-stream of its cleavage site. Flanking the target ssDNA with a pair of such DNAzymes generates a precursor ssDNA amplifiable by pseudogene-recombinant bacteriophage, which subsequently releases the target ssDNA in large quantities after efficient auto-processing. PECAN produces ssDNA of virtually any terminal bases and compositions with >98.5 % purity at the milligram-to-gram scale. We demonstrate the feasibility of using PECAN ssDNA for RNA in situ detection, homology-directed genome editing, and DNA-based data storage.
合成单链（ss）DNA是生命科学和材料科学的基石，然而，合成DNA的纯度、数量、长度和可定制性在各种应用中仍然受到限制。在此，我们展示了PECAN，即由脱氧核酶（DNA酶或脱氧核糖酶）辅助的双端切割技术，该技术能够以单碱基精度大规模生产任意序列（长达7000个核苷酸，即nt）的ssDNA。PECAN技术的核心是两类新鉴定的脱氧核酶，每一类在其切割位点上游或下游具有最低序列要求的情况下都能强烈地自我水解。在目标ssDNA两侧连接一对这样的脱氧核酶会产生一种可通过假基因重组噬菌体扩增的前体ssDNA，该前体ssDNA随后在高效自动加工后大量释放目标ssDNA。PECAN在毫克到克的规模上可生产几乎任何末端碱基和组成的ssDNA，纯度>98.5%。我们证明了使用PECAN ssDNA进行RNA原位检测、同源定向基因组编辑和基于DNA的数据存储的可行性。
