Tth Argonaute (TtAgo)

Catalog #	Concentration	Size	List Price	Quantity	Your Price
M0665S		50 pmol	$120.00		$108.00

Catalog #	Size	List Price	Your Price
M0665S	50 pmol	$120.00	$108.00

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 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.

Reagents Supplied

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

M0665S -20

	Tth Argonaute (TtAgo)	M0665SVIAL	-20	1 x 50 pmol	1 µM
	ThermoPol^® Reaction Buffer	B9004SVIAL	-20	1 x 1.5 ml	10 X

An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

Companion Products

Notes

Mg²⁺ is present in 1X ThermoPol^® Reaction Buffer as 2mM Mg_SO₄ 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.

References

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.

Protocols

Usage Guidelines

GUIDElines for optimization of Tth Argonaute (TtAgo) reactions

FAQs

Citations

Additional Citations

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

Quality Control Assay

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety Data Sheets

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