NEB TV Ep. 31 - What are Argonaute Proteins?

In this video, learn more about argonautes, including how these nucleic acid-guided endonucleases differ from Cas enzymes and potential applications.

Script

Deana Martin:
Welcome to NEB TV. Today I am joined by Eric Hunt who is a scientist in our applications and product development group. Hi Eric.

Eric Hunt:
Hi.

Deana Martin:
And we are talking about Argonautes. Eric, could you start off by telling us what an Argonaute is?

Eric Hunt:
Yeah. So the prokaryotic Argonaute is a nucleic acid guided endonuclease, which means unlike normal endonucleases where you'll have a cutting at a lot of random locations, prokaryotic Argonaute uses a short nucleic acid guide to bring its activity to a very specific sequence in a substrate that's complimentary to that guide.

Deana Martin:
Okay. And how do they differ from Cas enzymes?

Eric Hunt:
So they're actually a very different class of enzyme from Cas enzymes. Cas enzymes have two active sites, so they're able to cut in two places on a double-stranded substrate. They still use a nucleic acid guide. So in the case of something like Spy Cas9, which everyone's very familiar with, uses an RNA guide to bring its activity to that location. And then each active site cuts each strand of that double-stranded substrate.

Eric Hunt:
With a prokaryotic Argonaute, that single guide is only cutting opposite of its complimentary sequence. So there's only one cut. So in the case of a double stranded substrate, you'd just be using it like a nickase. Almost like the D10A nickase Cas9 that we sell.

Eric Hunt:
For the guide portion of it, we sell a sgRNA kit to be able to simplify the synthesis process of a Cas9 guide. It's even easier with an Argonaute, they just use short DNA or RNA guides. In the case of Thermus thermophilus Argonaute, which we just launched. It's a short DNA guide. So you could order them from your favorite vendor and just add a phosphate to it and you have a guide that's ready to use.

Deana Martin:
And what kind of substrates do Argonautes target?

Eric Hunt:
So depending on the species that the Argonaute is from, they can target DNA or RNA substrates and they can use DNA or RNA guides. Really, there's four big major categories that they would fall into. Some of them have some cross-reactivity between those different categories. For example, Thermus thermophilus Argonaute is a DNA guided DNA endonuclease, but it also has some activity on RNA substrates as well.

Deana Martin:
Okay. Will they cut in a specific location?

Eric Hunt:
Yes. So Thermus thermophilus Argonaute cuts directly across from positions 10 and 11 in the sequence that's complimentary to the guide.

Deana Martin:
We have an animation that overviews how this Argonaute works. So we'll take a look at that.

Eric Hunt:
Great.

Narrator:
Prokaryotic Argonautes are nucleic acid guided endonucleases involved in prokaryotic cellular defense against foreign genetic elements. When provided with synthetic nucleic acid guides, prokaryotic Argonautes can be used as programmable nucleases.

Narrator:
Argonautes from different prokaryotic species may utilize either DNA or RNA guides to target DNA or RNA substrates, leading to many potential combinations of guide and substrate preferences.

Narrator:
Prokaryotic Argonaute proteins are typically comprised four domains: the MID domain, the PAZ domain, the PIWI domain, and the N domain.

Narrator:
The MID domain of prokaryotic Argonaute binds a short single stranded oligonucleotide guide, which is typically 16 to 18 nucleotides in length. The guide is often five-prime phosphorylated, so some Argonautes can utilize guides with other five-prime chemical modifications.

Narrator:
The PAZ domain is responsible for holding the three-prime end of the guide while the Argonaute searches for a matching sequence. The prokaryotic Argonaute guide complex searches for a complimentary sequence on the target strand when it match is found, the three-prime end is released to allow for complete base pairing between the guide and substrate. This activates prokaryotic Argonaute endonuclease activity in the PIWI domain, which contains a metal dependent RNase H-like active site. A break is created in the phosphodiester backbone of the complimentary substrate nucleic acid. In the case of double stranded substrates, a break is only created in the strand, which is complimentary to the guide nucleic acid.

Narrator:
The N domain is thought to then act as a wedge that helps release the target DNA. Thermus thermophilus Argonaute, also known as TtAgo is the first commercially available prokaryotic Argonaute from NEB. TtAgo is a thermal stable prokaryotic Argonaute, which utilizes short five-prime phosphorylated single stranded DNA guides to target and cut DNA substrates at temperatures between 65 and 85 degrees Celsius.

Narrator:
For more information on TtAgo, please visit neb.com/M0665.

Deana Martin:
Eric, how does one go about designing guides?

Eric Hunt:
So the design process for a guide is pretty easy. We've worked pretty hard to put up a lot of FAQ and a few guidelines to help uses along. But they're simply short 16 to 20 nucleotide guides. We recommend 16 to 18 nucleotides for the best activity.

Eric Hunt:
They're single stranded DNA oligos with a five-prime phosphate. So we have a couple of different methods. You could order them directly with the phosphate modification already present or we also have some methods if you want to do some high throughput screening or a little bit more economical way to make them. If you're not really sure what you want to target right away, you could just order unmodified guides and then use our T4 PNK polynucleotide kinase to add a five-prime phosphate to those guides. Actually, if you just heat it back to activate the enzyme, you can use it directly right after that. So it's a pretty simple way.

Deana Martin:
Can Argonautes be used in vivo?

Eric Hunt:
Currently, no. The most well-studied prokaryotic Argonautes, as we said, are thermophiles and that just makes the temperatures that they function at incompatible with, for example, an in vivo gene editing application, like Cas9 really excels at. But there's been some recent reports of new mesophilic prokaryotic Argonautes and the hope is that someday we'll discover one that could potentially be an alternative tool to kind of add to that endonuclease toolkit for gene editing.

Deana Martin:
Right, right. So what applications can you use the thermophilic Argonautes for?

Eric Hunt:
Currently, some of the applications are usually involved in diagnostics, either directly or indirectly. Typically, something like an enrichment application where you're trying to get rid of a bunch of wild type sequences to really enrich for a rare sequence so you can use the Argonautes to deplete that. There's also been a couple of publications where the Argonaute's been used directly in sort of a biosensor-like fashion to be able to detect a specific sequence. We're focusing on these applications, but we're really excited to see what our customers come up with because they're some of the most inventive researchers out there.

Deana Martin:
That's true, that's true. Could you tell us a little bit about how NEB discovered this enzyme?

Eric Hunt:
Yeah. I think the story of TtAgo holds pretty true to this vision that our founder Don Comb had for the company to be a company of scientists for scientists.

Eric Hunt:
There's a lot of times where a researcher in our research department will say, "Wow, this is really cool. Check this out." Our CSO, Rich Roberts started this enzymes for innovation program where we're sort of able to release these cool enzymes and kind of share that, "Wow, isn't this cool moment with our customers." And TtAgo's one of those enzymes we don't really have an exact application for it per se, but we're excited to share it with everybody.

Deana Martin:
And see what they come up with.

Eric Hunt:
Yeah. See what they come up with.

Deana Martin:
Right, right. Will any be releasing additional Argonautes in the future?

Eric Hunt:
Argonautes remain very interesting class of enzymes for us and we're still actively researching them. I couldn't say exactly what the next Argonaute would be, but yeah, we're still interested in them. So stay tuned.

Deana Martin:
That's exciting. That's great. If a customer wants to learn more about these Argonautes, where should they go?

Eric Hunt:
So our website on the Thermus thermophilus Argonaute page is full of information that we've added. We're also still looking to actively publish a lot of the work that we've done to be able to share some of this data and methods with researchers who are interested in getting in this field. But I would say the product webpage, we have some videos up. We have some guidelines for designing guides and some data that people might find interesting as well.

Deana Martin:
That's great.

Eric Hunt:
It's a good place to start.

Deana Martin:
All right. Well, thanks so much for joining me today.

Eric Hunt:
Thank you.

Deana Martin:
We're excited to see what you do next.

Eric Hunt:
Yeah.

Deana Martin:
All right. Well, if you have any suggestions for future episodes, please let us know.


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