Szokoli Deni, Mutschler Hannes
Biomimetic Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 4a, Dortmund 44227, Germany.
Nucleic Acids Res. 2025 Jan 11;53(2). doi: 10.1093/nar/gkae1224.
Group II introns are ancient self-splicing ribozymes and retrotransposons. Though long speculated to have originated before translation, their dependence on intron-encoded proteins for splicing and mobility has cast doubt on this hypothesis. While some group II introns are known to retain part of their catalytic repertoire in the absence of protein cofactors, protein-free complete reverse splicing of a group II intron into a DNA target has never been demonstrated. Here, we demonstrate the complete independence of a group II intron from protein cofactors in all intron-catalyzed reactions. The ribozyme is capable of fully reverse splicing into single-stranded DNA targets in vitro, readily hydrolyzes DNA substrates and is even able to unwind and react with stably duplexed DNA. Our findings make a protein-free origin for group II introns plausible by expanding their known catalytic capabilities beyond what would be needed to survive the transition from RNA to DNA genomes. Furthermore, the intron's capacity to react with both single and double-stranded DNA in conjunction with its expanded sequence recognition may represent a promising starting point for the development of protein-free genomic editing tools.
II 组内含子是古老的自我剪接核酶和逆转座子。尽管长期以来人们推测它们起源于翻译之前，但它们对内含子编码蛋白进行剪接和移动的依赖对这一假说提出了质疑。虽然已知一些 II 组内含子在没有蛋白质辅因子的情况下仍保留部分催化功能，但 II 组内含子在无蛋白质条件下完全反向剪接进入 DNA 靶点的情况从未得到证实。在此，我们证明了 II 组内含子在所有内含子催化反应中完全不依赖蛋白质辅因子。该核酶能够在体外完全反向剪接进入单链 DNA 靶点，能轻易水解 DNA 底物，甚至能够解开稳定的双链 DNA 并与之反应。我们的研究结果通过扩展 II 组内含子已知的催化能力，使其超越从 RNA 基因组过渡到 DNA 基因组所需的能力，从而使 II 组内含子无蛋白质起源变得合理。此外，该内含子与单链和双链 DNA 反应的能力及其扩展的序列识别能力，可能代表了开发无蛋白质基因组编辑工具的一个有前景的起点。
