Welcome to NEB TV. Today I am
joined by Eric Hunt who is a scientist
in our applications and product development group.
Hi, Eric.
And we're talking about Argonautes. So,
Eric, could you start off by telling us what an Argonaute is?
Yeah. So a prokaryotic Argonaute is
a nucleic acid guided endonuclease, which
means, unlike normal endonucleases,
where you'll have
cutting in 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 complementary to that guide. Okay
and how do they differ from Cas enzymes?
So they're actually a very different class of enzyme
from Cas enzymes. Cas enzymes have
two active sites ,
so they are able to cut in two places
on a double-standard 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 the double-stranded
substrate.
With a prokaryotic Argonaute,
That single guide is only cutting opposite
of its complementary sequence.
So there's only one cut. 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.
So 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 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. And
what kind of substrates do Argonautes target?
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. So really, there's four
big major categories that they would fall into,
and some of them have some crossreactivity between
those different categories. So, for example,
Thermus thermophilus Argonaute,
it is a DNA guided DNA endonuclease, but
it also has some activity on RNA
substrates as well. Okay, and will they
cut in a specific location? Yes.
So, Thermus thermophilus Argonaute cuts
directly across from Positions 10 and 11
in the sequence that's complementary to the guide.
So we have an animation that overviews how
this Argonaute works, so we'll take a look at that.
Prokaryotic Argonautes are nucleic acid
guided endonucleases involved in
prokaryotic cellular defense against foreign
genetic elements. When provided with synthetic
nucleic acid guides, prokaryotic Argonatues
can be used as programmable nucleases.
Argonauts 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.
Prokaryotic Argonaute proteins are typically
comprised of four domains, the MID domain,
the PAZ domain, the PIWI domain
and the N domain.
The MID domain of prokaryotic Argonaute
finds a short, single-stranded oligonucleotide
guide, which is typically 16 to 18
nucleotides in length.
The guide is often five prime phosphorylated,
though some Argonautes can utilize guides with
other five prime chemical modifications.
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 a 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 complementary substrate
nucleic acid.
In the case of double-stranded substrates, a
break is only created in the strand which is
complementary to the guide nucleic acid.
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 thermostable
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.
For more information on TtAgo,
please visit neb.com/M0665
So, Eric, how does one go about designing guides?
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 users along. But
they're simply short 16
to 20 nucleotide guides. We recommend
16 to 18 nucleotides
for the best activity.
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
use our T4 PNK
polynucleotide kinase to add a five prime phosphate
to those guides. And actually, if you
just heat inactivate the enzyme, you can use
it directly right after that, so it's a pretty simple
way. So can Argonautes be used
in vivo?
So 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, like 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.
That kind of add to that
endonuclease toolkit for gene editing. Right.
Right. So what applications can you use
the thermophilic Argonautes for? So 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 off
a bunch of wild type sequences to really enrich
for a rare sequence.
So you could 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.
You know, we're focusing on these applications,
but we're really excited to see
what our customers come up with, cause they're some of most
inventive researchers out there. That's true. That's
true. So could you tell us a little
bit about how NEB discovered this enzyme? *NEB commercialized TtAgo, first crystalized by Y. Wang et al. (2008) Nature, 456, 209-213, 921-926.*
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.
There's a lot of times where
a researcher in our research
department will say, 'Wow, this is this is really cool.
Check this out.'
Our CSO, Rich Roberts
started this enzymes for innovation
program. We're sort of able to
release these cool enzymes and kind of share that
'Wow, isn't this cool' moment with our customers.
TtAgo's one of those enzymes and
we don't really have an exact application for
it, per se, but
we're excited to share it with with
everybody. And see what they come up with? That's right.
Right, and will NEB be releasing
additional Argonautes in the future? So
Argonautes remain a
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 we're still interested.
That's exciting, too. That's great.
So if a customer wants to learn more about
these Argonautes, where should they go? So our
website.
On the Thermus thermophilus Argonaute page
is full of information
that we've added. And 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, you know the product web
page. We have some videos up. We have
some guidelines for designing guides and
some data
that people might find interesting as
well. That's great. It's a good place to start. All right,
well, thanks so much for joining me today. I'm excited
to see what you do next.
All right.
Well, if you have any suggestions for future
episodes, please let us know.