Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute

Daan C. Swarts, Malwina Szczepaniak, Gang Sheng, Stanley D. Chandradoss, Yifan Zhu, Elizabeth M. Timmers, Yong Zhang, Hongtu Zhao, Jizhong Lou, Yanli Wang, Chirlmin Joo, John van der Oost

Research output: Contribution to journalArticlepeer-review

79 Scopus citations


Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5′ end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5′ end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

Original languageEnglish
Pages (from-to)985-998.e6
JournalMolecular Cell
Issue number6
StatePublished - 16 Mar 2017

Bibliographical note

Publisher Copyright:
© 2017 Elsevier Inc.


  • DNA chopping
  • RNA interference
  • TtAgo
  • ago
  • guide generation
  • guide loading
  • pAgo
  • prokaryotic argonaute
  • siDNA
  • small interfering DNA


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