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Thermus thermophilus argonaute (TtAgo) is a programmable DNA-endonuclease which requires a short 5’-phosphorylated single-stranded DNA guide to target its activity to a specific corresponding sequence on a substrate. TtAgo introduces one break in the phosphodiester backbone of the complementary substrate sequence.
Short 16–18 nucleotide 5′-phosphorylated ssDNA guides are cost effective and can be phosphorylated with NEB T4 Polynucleotide Kinase
Guide/target sequence selection is not limited by the requirement of an adjacent sequence motif
Highly active on ssDNA and most dsDNA substrates (generates a nick in dsDNA substrates), with mild activity on ssRNA substrates
TtAgo is a prokaryotic argonaute from the Gram-negative, thermophilic bacterium Thermus thermophilus which functions as a DNA-guided endonuclease when provided with a 16–18 nucleotide long 5′-phosphorylated single-stranded DNA oligonucleotide guide. The RNase H-like active site of TtAgo requires divalent Mg2+ (or Mn2+) (See product usage note #1) metal ions for the reaction to occur and cleaves a complementary substrate between the bases corresponding to positions 10 and 11 of the DNA guide.
TtAgo mechanism of action
TtAgo is active from 65–85°C (See product usage note #2) and exhibits high activity on single-stranded DNA, moderate to high activity on double-stranded DNA, and mild activity on single-stranded RNA substrates. TtAgo does not introduce double-strand breaks in double-stranded DNA substrates when provided with a single guide, and instead only introduces a nick in the strand complementary to the guide. Activity is not observed on double-stranded RNA substrates. The corresponding 5ʹ and 3ʹ ends of cleavage products are compatible with downstream ligation.(1) It has been demonstrated that in some cases, the addition of thermostable single-stranded DNA binding protein (ET SSB, NEB #M2401) and/or a thermostable helicase can aid in the cleavage of some dsDNA substrates.
In the absence of a guide, the apo-form of TtAgo can degrade dsDNA through a process known as “chopping,” which is thought to be the mechanism by which TtAgo naturally generates guides from invading or foreign DNA.(2) To prevent this non-specific activity, guide should be kept in an approximately 5X molar excess over TtAgo. Additionally, a pre-loading step can be performed in which guide is incubated with TtAgo at ~75°C for 10-30 mins to form the nucleoprotein complex prior to substrate addition. Please see the Protocols, Manuals & Usage section for more details.
TtAgo is recommended for in vitro applications.
For larger volume or higher concentration requirements, customized and bulk packaging is available by purchasing through the Customized Solutions department at NEB. Please contact custom@neb.com for further information.
Product Source
Thermus thermophilus argonaute (TtAgo) is purified from an E. coli strain that carries a cloned gene from the Gram-negative, thermophilic bacterium Thermus thermophilus which is expressed as a recombinant N-terminal 6X His-tagged fusion.
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Mg2+ is present in 1X ThermoPol® Reaction Buffer as 2mM MgSO4 and is suitable for the majority of applications. Mn2+ can also be utilized by TtAgo, however, it should be used at approximately ten-fold lower concentrations as high concentrations of Mn2+ can become inhibitory to TtAgo activity and can lead to non-specific substrate degradation at high temperatures, especially with RNA substrates. For more information, please refer to the FAQ and GUIDElines for optimization of Tth Argonaute (TtAgo) reactions.
TtAgo is active from 65–85+°C. Routine reactions should be carried out between 75–80°C. Activity decreases sharply below 70°C. See the GUIDElines technical reference for more details.
Single-stranded DNA guide oligonucleotides should be 16–18 nucleotides in length and contain a 5ʹ-phosphate, which can be added post-synthesis with NEB T4 Polynucleotide Kinase.
Guide oligonucleotide should be used at 5X molar excess to TtAgo.
Diluent B can be used to dilute TtAgo. Dilutions should be stored at -20°C.
Hunt, E. A., Evans Jr, T. C., & Tanner, N. A. (2018). Single-stranded binding proteins and helicase enhance the activity of prokaryotic argonautes in vitro. PloS One. 13 (8), e0203073.
Swarts, D., Szczepaniak, M., Sheng, G., Chandradoss, S., Zhu, Y., & Timmers, E. et al. (2017). Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute. Molecular Cell.. 65 (6), 985-99.
Wang, Y., Sheng, G., Juranek, S., Tuschl, T., & Patel, D. (2008). Structure of the guide-strand-containing argonaute silencing comple. Nature. 456(7219), 209-213.
Swarts, D., Jore, M., Westra, E., Zhu, Y., Janssen, J., & Snijders, A. et al. (2014). DNA-guided DNA interference by a prokaryotic Argonaute. Nature. 507(7491), 258-261.
Sheng, G., Zhao, H., Wang, J., Rao, Y., Tian, W., & Swarts, D. et al. (2013). Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proceedings Of The National Academy Of Sciences. 111(2), 652-657.
Lin, Q., Han, G., Fang, X., Chen, H., Weng, W. and Kong, J. (2022) Programmable Analysis of MicroRNAs by Thermus thermophilus Argonaute-Assisted Exponential Isothermal Amplification for Multiplex Detection (TEAM). Anal Chem; DOI: 10.1021/acs.analchem.2c01945
Hunt E.A., Tamanaha E., Bonanno K., Cantor E.J. and Tanner N.A. (2021) Profiling Thermus thermophilus Argonaute Guide DNA Sequence Preferences by Functional Screening. Front Mol Biosci; 8, 8:670940. DOI: 10.3389/fmolb.2021.670940
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