School of Basic Medical Science and Clinical Pharmacy, China Pharmaceutical University, Nanjing 210000, China.
Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.
Nucleic Acids Res. 2020 Nov 18;48(20):e117. doi: 10.1093/nar/gkaa843.
Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.
在这里，我们描述了一种 flap endonuclease 1 (FEN1) 加发夹 DNA 探针 (hpDNA) 系统，命名为 HpSGN 系统，用于无序列限制的 DNA 和 RNA 编辑。HpSGN 系统的紧凑尺寸使其成为体内递送应用的理想候选者。体外生化研究表明，与我们之前报道的 SGN 系统相比，HpSGN 系统需要更少的核酸酶来切割 ssDNA 底物，效率约提高 40 倍。此外，我们证明 HpSGN 系统可以在体外有效地切割不同的 RNA 靶标。HpSGN 系统分别在细菌和人类细胞中切割基因组 DNA 的效率约为 40%和 20%，并在人类细胞中敲低特定的 mRNA 水平约为 25%。此外，在体外和体内，HpSGN 系统对发夹旁位置的单个碱基错配都很敏感。总的来说，这项研究表明，开发 HpSGN 系统作为一种小型、有效和特异性的编辑工具，用于在无序列限制的情况下操纵 DNA 和 RNA 具有潜力。
