FAQ: How can I reduce non-specific endonuclease activity with TtAgo?

Non-specific activity can be observed with double-stranded substrates, as TtAgo exhibits a degradative activity in its apo-form (in the absence of a guide), known as “chopping” [Swarts, et al. 2017]. This argonaute activity is thought to be involved in the creation of new guides for cellular defense, but can be problematic in vitro. We have observed that some guide sequences, while functional in guiding TtAgo endonuclease activity, may not be efficiently loaded into the argonaute. Thus, the “chopping” activity can take preference over the guided activity. In these situations, we recommend pre-loading the guide with TtAgo before attempting a reaction. Mix the guide and argonaute in 1X NEB ThermoPol® Reaction Buffer and incubate at 75°C for 10 minutes. We recommend using guide in a 3–5 times molar excess over TtAgo to reduce non-specific activity. We have also observed that reducing the reaction temperature and (to a lesser extent) titrating the amount of magnesium in the reaction can impact the amount of non-specific activity observed.