Product Class: Other

Tth Argonaute (TtAgo)
recombinant dil_B No


Catalog #M0665

Product Introduction

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
  • Recommended for in vitro applications
  • Refer to GUIDElines for optimization of Tth Argonaute (TtAgo) reactions

Product Information

Description

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 requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact the Customized Solutions team 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.
This product is related to the following categories:
Argonautes Products,
Programmable Nucleases Products,

Properties & Usage

Diluent Compatibility

Storage Buffer

10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

No

Product Notes

  1. 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.
  2. 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.
  3. 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.
  4. Guide oligonucleotide should be used at 5X molar excess to TtAgo.
  5. Diluent B can be used to dilute TtAgo. Dilutions should be stored at -20°C.

References

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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, Manuals & Usage

Protocols

  1. Protocol for generating break in phosphodiester backbone with Tth Argonaute (TtAgo, NEB #M0665)
  2. Protocol for creating 5′-Phosphorylated Guides from Unmodified Oligonucleotides for use with TtAgo (NEB #M0665)
  3. High-throughput protocol for phosphorylation of guide oligonucleotides in 96-well PCR plate format (NEB #M0665)

Usage & Guidelines

Application Notes

FAQs & Troubleshooting

FAQs

  1. Can I prepare functional guides for TtAgo from unmodified synthetic oligos?
  2. Some guides do not seem to be working as well as others with TtAgo. What can be done?
  3. How can I reduce non-specific endonuclease activity with TtAgo?
  4. Can I store TtAgo with a guide “loaded?”
  5. Is TtAgo active in other NEB buffers?
  6. Is TtAgo active at other temperatures?
  7. How much Mg2+ [magnesium(II)] should be included in a typical reaction?
  8. What if I want to use TtAgo with Mn2+ [manganese(II)] instead of Mg2+ [magnesium(II)] as provided in ThermoPol® Reaction Buffer?

Quality, Safety & Legal

Quality Assurance Statement

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 DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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