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ELIZABETH NOLAN: Where we left off yesterday

was beginning to discuss methods for unnatural amino acid

incorporation into proteins using the ribosome.

And the methodology that was introduced

and where we need to continue today

is the Schultz method of using the native ribosome

to play some tricks and get unnatural amino acids

into proteins.

So we'll work through this further to show

how the rest of the machinery was generated

and then we'll consider some of the limitations

and some of that came up in questions last time.

And then we'll close with a discussion

of one strategy that's a different strategy that

uses actually an orthogonal ribosome, which

is really, really neat here.

So where we left off last time in terms of the Schultz Method

was that we needed a unique codon

for the unnatural amino acid, right?

And a stop codon was reassigned.

So TAG or Amber stop.

And the other thing that we need that we'll discuss now

involves the requirement of an orthogonal tRNA

and aminoacyl-tRNA synthetase pair that

can be used in this method.

So the question is, where does this come from?

So where do we get a tRNA and an aaRS

that can be used for this unnatural amino acid

of interest.

And one way to think about this in terms of a search

is to think about different tRNAs and aaRS

from different organisms.

And so what's found if tRNAs are compared

between bacteria, eukaryotes, [INAUDIBLE],,

there's evolutionary divergence.

And so can that evolutionary divergence

be taken advantage of?

And effectively, is it possible to find

some tRNA and its aminoacyl-tRNA synthetase

from one organism that's orthogonal to the corresponding

tRNA and aminoacyl-tRNA synthetase

in the organism of interest.

So effectively, if we want to use E. coli,

we want to find a pair from another organism that's

completely independent of the endogenous E. coli machinery.

So what does this mean?

A lot of trial and error was done

to identify a pair from another organism.

And where they ended up finding one is from a methanogen.

So methanococcus jannaschii here.

OK, and this initial pair was for tyrosine.

And so, there's some features of this pair that

are noteworthy to bring up.

So first if we think about the aminoacyl-tRNA synthetase here.

This one has an unusual feature.

So when we discuss these aaRS, remember

we discussed the mechanism and we also

discussed what happens if a wrong amino acid is selected.

And we learned that they have editing function

and that there's editing domains.

What also came up in those discussions

is that we need to take every one of these enzymes

as a case-by-case basis.

And as it turns out, this particular enzyme

does not have an editing domain.

So then the thing to think about is,

why would that be useful from the standpoint of incorporating

an unnatural amino acid?

So what's the benefit there?

So what does this editing domain do?

AUDIENCE: It's one less thing to have

to fix if you're assuming that the editing domain would

recognize and hydrolyze an unnatural amino acid

that you put in even if you got the binding site to recognize

it, or the first binding site to recognize it.

ELIZABETH NOLAN: Exactly right.

There's no deacylation happening, so no hydrolosis.

So it's just more likely this unnatural amino acid can

be a successful substrate and there's

less engineering that has to be done in terms

of modifying the enzyme here.

So, another point in terms of--

they found that it does not acylate E. coli tRNA.

OK, and that's important for trying to use this in E. coli

here.

What's the potential problem?

So is this going to be specific for the unnatural amino acid

of interest?

No way, right?

Unlikely at least, and depending on what type

of a natural amino acid you're thinking about,

it may definitely be a no way.

So there's some experimental work

to do to make this specific for the unnatural amino acid

of interest, which means there has to be some mutagenesis

and selection, which we're not going

to talk about in detail here.

So what about the tRNA?

So we need to think about the tRNA structure right

and how tRNAs interact with aaRS, right?

And recall, we had in an earlier lecture one

example of a crystal structure of this complex.

And we saw there's many positions where they interact.

What was known in this system here is that they figured--

OK, and also keep in mind, just backing up a minute,

this tRNA, as we know, based on this nomenclature

has an anticodon for tyrosine.

So that's going to have to be mutated

to be the anticodon on the Amber stop

in order to use in this method.

Right?

So this is going to have to be mutated

to give us tRNA(CUA) where this is indicating

the anticodon here.

So that mutation, we don't want that mutation

to disrupt the interaction between the tRNA

and aminoacyl-tRNA synthetase, right?

And it turns out there were minimal interactions

in that area for the native system.

So the thinking was that these mutations

could be tolerated here.

So with that said, what is the potential problem

with this tRNA?

So if we want to put this tRNA into E. coli,

it can't be recognized by any of the E. coli aaRS.

So all of these recognition issues come up.

So, here again with another example,

where there needed to be some mutagenesis and selection

to prevent interactions between this tRNA

from the methanogen and the aaRS of E. coli.

And effectively what they did is to pick 11 positions

on the tRNA, which I'll just chart out.

OK, so here's our tRNA.

Here's our CUA anticodon here.

And effectively these ends are positions where they randomized

and did mutagenesis here.

So they identified these 11 positions.

OK?

And these 11 positions do not interact

with the aaRS here of the pair.

So the idea is to maintain this interaction,

but prevent any interaction of this tRNA with E. coli

machinery here.

So effectively, they used a method

called directed evolution to do selection.

And what might happen out of that,

imagine you have some large pool of mutant tRNA,

what might happen?

So here OK, so the end result is that the tRNA

might be non-functional.

Right?

So the mutation was not helpful.

OK?

It might be non-orthogonal, meaning

that it's recognized by the endogenous E. coli machinery

or it may be orthogonal here.

OK, so recognized only OK?

And so this is what needs to be selected for here.

And so assays need to be done that allows these

to be differentiated here.

So the end result is an orthogonal pair.

But the point is, you can't just take this pair

from the other organism.

It needs to be further modified.

So where does that put us in terms

of the cartoon we saw yesterday without some of these details?

So here we have the tRNA that has this amber anticodon.

So that's our orthogonal tRNA.

We have an unnatural amino acid that's

able to get into the organism of interest.

And we have the orthogonal tRNA synthetase.

So these give us this aminoacyl-tRNA

with the unnatural amino acid.

And then that can be incorporated into the A

site of the ribosome.

Right so this is a case where we have a plasma DNA.

Here's the gene of interest in red.

And somewhere in that gene, a stop codon

has been placed to allow for incorporation

of this unnatural amino acid somewhere

within the polypeptide chain as shown here.

So if we think about the scope of this methodology,

where does this take us?

So, it's quite broad.

This type of work has been applied beyond E. coli,

so in yeast and mammalian cells.

At present, there is many, many different unnatural amino acids

that can be incorporated and it's used by many labs.

So that's something to keep in mind.

If you're developing a new method,

you'd really like other folks in other labs

to be able to use your method.

There's a lot of troubleshooting to do experimentally

to get it up and running.

And Joanne's a wonderful person to talk about that

if you're curious for details.

Just some amino acid scope, and you

know what maybe we could do.

So these are some earlier examples

of unnatural amino acids that can be incorporated.

And what are some of the neat things?

If we look just here for example, there's an azide.

Why might we want an azide?

AUDIENCE: Click chemistry.

ELIZABETH NOLAN: Yeah, click chemistry, right.

Some chemistry that could be done after protein expression

or maybe in a cell.

Here we have a benzophenone.

So they're useful for cross linking experiments

and we'll likely talk about benzophenone cross linking

in recitation five in detail.

We see some sugars here.

This is the damsel group.

That's a fluorophore.

So there's many possibilities here.

Just looking at these molecules, what's something

similar about all of them?

We think about them compared to a native amino acid.

AUDIENCE: I was just that they're small.

ELIZABETH NOLAN: OK, they're quite small.

AUDIENCE: It's a kind of modified tyrosine.

It will have some sort of benzo group that's modified.

ELIZABETH NOLAN: So they're sort of

phenylalinine or tyrosine like, right?

And does that make sense from the standpoint

of using this machinery initially?

Yes, and you can imagine looking for other pairs

to put in other types of unnatural amino acids.

So that's reflective there.

Just as some further examples, So this

is another example of using an unnatural amino acid that can

be useful for click chemistry.

And I picked this in part, for one, this unnatural amino acid

looks very different than the ones we saw on the prior slide.

But there's aminoacyl-tRNA synthetase

and the tRNA for this alkyne.

And so you can imagine expressing a protein with this

at a specific location.

And then after the fact, clicking on a molecule

like this fluorophore here.

So just thinking about this process,

why maybe was this put on later rather than in the cell?

AUDIENCE: Do you mean clicking it on or synthesizing--

or putting that whole thing on?

ELIZABETH NOLAN: Yeah, as you can imagine someone

could have thought, rather than clicking this