Introduction to Chemical Biology 128. Lecture 05. Non-Covalent Interactions, DNA.

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>> My name is Javert.
Was that at least as good as Russell Crowe?
OK, so we're ready to get started.
First a quick quote, "Chemistry is to biology,
what notation is to music."
To me this really grabs at the essence of chemical biology
in the sense that the notations
on a musical scale allow creativity.
They allow other reformers to interpret the works in new ways
and give the work context.
Chemistry does that in biology.
Chemistry gives us an opportunity for us
to be creative about biology and invent new ways
of thinking about biology.
It's sort of the underlying basis at the level of atoms
and bonds as I keep saying, for biology,
and to me in some way this really captures what we're
trying to do in this class.
OK. So this week, where it's already week 3
which is amazing-- Oh, hang on.
OK, so it's week 3 so we're up to chapter 3 and we're going
to be talking about DNA.
Our knowledge of DNA was really set in place
by the people in front of you.
These are the giants really in the field of structural biology
who determined structures of DNA in the 1950s.
This includes the great Rosalind Franklin whose very accurate
x-ray diffraction structures and her pictures
of the x-rays diffracting off the fibers of DNA set
in motion the determination of the structure.
She was working with Maurice Wilkins and two physicists,
Francis Crick and Jim Watson went
on to solve the structure of DNA.
And as we'll see in a moment,
really one of their key insights was at the level of atoms
and bonds in the sense that they discovered interesting
tautomerization of the DNA bases that made it possible
to have what we now call Watson-Crick base pairing
between the strands of DNA.
Getting a little ahead of myself but that's where we're going
in the next week or so.
So we're going to be finishing
up non-covalent interactions then talking
about DNA structure, DNA property
and finally DNA reactivity of small molecules.
This is a large chapter.
We have a lot to talk about, so bear with me.
Things are going to go not faster it's going
to be the same speed, but we're going to gloss
through a few topics that are less important and when we do,
this means then that you can focus your reading
and your study just on the level of detail
that we're covering in the class.
OK. Some announcements, in the textbook, read chapter 3.
Again, skim concepts not presented in lecture,
don't get too worked up about them.
Chapter 3 problems, do all of the odd numbered and all
of the asterisked problems.
In addition, I want to encourage you
to get involved here at UC Irvine.
This is super important.
Many of you I know aspire to become physicians or scientists
or pharmacists or whatever it is that you aspire to do.
All those big plans require preparation,
they require some evidence that you've gone beyond the ordinary
and I want to encourage you to do this.
OK. One way to get involved is to look
around for opportunities to volunteer.
This is one that's run by my friend who is one
of the founders, it's called the Social Assistance Program
for Vietnam.
If you go to this website, there are opportunities to volunteer
to spend two weeks in Vietnam, in a rural part
of Vietnam administering medicine, you know.
You'll probably not be of course, you know,
drilling people's teeth and, you know, doing open heart surgery
but you will get a unique opportunity
to actually see those types of things happening
and that's really important if you aspire
to that kind of career.
It provides evidence that you're qualified, that you're committed
and that you're someone who is altruistic.
All of those things professional and graduate schools look
for in your application.
You need to be doing those things now.
OK. And I'm on your side on this.
OK. I will help you get-- find those opportunities.
I'll bring them to your attention like this one.
And if there's something in particular that I can do
to connect you with, let me know
and I'll do my very best on your behalf.
OK. Along those lines, our laboratory always has openings
for talented undergraduates.
It's competitive but you have a chance to participate
at the full level of a graduate student.
Undergraduates in our laboratory are doing actual science.
They're publishing papers with us.
They're making discoveries and they're participating
as full members of the team.
OK. Here's how you apply,
send me a paragraph describing your career goals
and how research
in our laboratory would advance those career
and educational goals.
In addition, send me a copy of your college level transcripts.
This includes any transcripts at community colleges
if you're transferring.
Many of my best students are transfer students.
Send me those transcripts as well
and also send me three names and email addresses of TAs
who know you well in lab sections.
OK? And I'm going to email them and I'm going
to ask them what was this person like in the laboratory?
Were they, you know, the first one out of the room?
Were they last one out of the room?
Did they, you know, follow you
around the laboratory asking you,
"Does this look pink, does this look pink?"
or were they pretty independent, OK?
So I'll find out about that sort of thing
and then that's how I make a decision on who
to accept into the laboratory.
OK. And then of course the resume.
This is pretty standard, if you're interested
in doing research here at UC Irvine which I highly,
highly encourage you to do,
this is a pretty good way to go about it.
OK. This is an effective way to get noticed and to get
that job that you need.
OK. Any questions about these opportunities?
Why I think they're important and things like that?
OK. See me in office hours if there's something in particular
that you want from me and I'll try to hook you up.
OK, office hours this week, speaking of which.
Tomorrow I'll have my usual office hour,
2.45 to 3.45, the usual location.
Thursday I'll have my office hour 11 to 1, usual location.
In addition, Mariam will have her office hour Fridays 1:30
to 2.30.
And, Kritika, could you raise your hand?
So, Kritika is our new TA.
She'll be joining in the team.
And Kritika, does this time work for you, Tuesdays 2:30 to 3:30?
Good. OK. And she'll be having her office hours Tuesday.
So notice that we spread out our office hours
so that there's one everyday of the week except Monday
because I know you're very busy
on Monday doing all kinds of things.
I hope you're having fun yesterday.
But yeah, so everyday of the week there's an office hour,
they're staggered so they're at different times,
so you can have your questions answered.
And again, Kritika is a graduate student in my laboratory.
She knows this material as well as I do.
She's really smart.
You can go to her office hour and get an answer.
That's as good as an answer that I will give you.
OK? And for that matter you can also email the TAs
with your abundant questions.
OK. I'm looking at you where I can find that person, OK,
there's like one person in the class,
he send me 10 emails a day but, you know, I will do my best
but you can also email the TAs as well.
OK. Oh, along those lines,
I sent you an email saying don't send me book
or potential journal articles.
And the reason is I must do-- I open my inbox and I had like 15
of those and I got to the point where I was bouncing messages
because the inbox was so full.
So, if you send me those, I can't do very much with them,
OK, it might clog my box.
So what I propose we do is instead of you emailing me them,
instead bring them to my office hours,
bring them to Kritika's office hours or Mariam's office hours
and ask your questions then.
OK. Now the standard question I get asked is,
is this article appropriate?
And my answer to that is if you follow the guidelines,
it will be appropriate.
Now in addition, when you're writing your summary,
your report on the journal article, focus on the aspects
of the article that fit the definition of chemical biology.
OK. So a paper in cell for example is going
to be a very meaty paper, it's going to cover about 10 pages.
It's going to have, you know, eight or nine figures and some
of those figures, some of those experiments aren't exactly what
we will call chemical biology.
Don't focus on those.
Focus on the ones that are chemical biology related.
OK. Otherwise I don't know
that you know the definition of chemical biology.
OK. Any questions?
All right, guess what, we're heading into midterm season.
This is week 3, so week 4 is next week.
We will have a midterm next Thursday,
a week from this Thursday.
There will be a review session in advance
by the TAs, time to be announced.
Kritika will arrange for this.
The seating for the midterm will be assigned.
Mariam, you'll do the assignment.
It's really essential that you bring your UCI student ID.
We will check the IDs to make sure you're seated
in the right seat.
If you're not seated in the right seat, it will be treated
as an academic honesty infraction.
No notes, no calculators,
no electronic devices, you don't need them.
You're smart.
OK? Any questions about the upcoming midterm?
OK, now I know you want to know what will be on the midterm.
OK, so let me tell you, it will cover
through Tuesday's lecture one week from today
and so we will be about halfway
through chapter 4 on Tuesday, OK?
So plan to read through about halfway through chapter 4,
that's the chapter on RNA and that's where I expect to be
for Tuesday's lecture.
It's possible I might get behind but I'm going
to really try hard not to do that.
OK. All right, I will also post a practice midterm
to the website and you can use that along
with the discussion worksheets, the assigned problems as a guide
for what will be on the midterm.
OK? So the midterm will look very much like a compilation
of discussion worksheets, of assigned problems
and the practice midterm.
OK. And it will be about as long as the practice midterm as well.
So when the practice midterm comes out,
I'll post two versions.
One version will be blank, one version will be the key.
The blank version you should print out
and then give yourself an hour and 20 minutes and make sure
that you can handle it.
OK. And at the end of that,
then check your answers against the key.
But give yourself a real practice.
OK. That's pretty important, I think.
OK, so anyway, that's the plan, any questions
about the midterm coming up?
I know you will have lots of questions.
I look forward to hearing about them in my office hours.
All right, I want to go back to finish up our discussion
of non-covalent interactions and where we left off last time was
with charge-charge interactions.
I'm not ready to talk to you about interactions
between atoms that are uncharged.
OK. Neutral atoms that are interacting with each other.
These are described by a Lennard-Jones Potential
which is an equation
that describes how these neutral atoms would interact
with each other.
Another way of describing these neutral atoms,
another term that's used and probably one
that you encountered is London dispersion force.
OK. So when you have two say neon atoms that cozy up next
to each other, then they will interact
through a London dispersion potential or force
and that's what I'm describing here.
OK. So, it's just a couple of different ways
of saying the same thing.
This happens a lot in biology, not necessarily
between neon atoms but certainly between aliphatic side chains,
hydrophobic side chains in proteins, in interactions
with each other, interactions with lipids, at the--
a plasma membrane of the cell, and a whole host
of other non-covalent hydrophobic-hydrophobic
type interactions.
This turns out to be a very potent
and very strong force in biology.
OK. So we need to understand it better.
So the energy-- the potential energy
of a van der Waals interaction, yet another word to describe it,
is equal to-- is proportional to 1 over r
to the 12th minus 1 over r to the 6.
These terms, the sigma term deals with the diameter
in this epsilon ij, not so important, so let's ignore that.
Let's focus in on the 1 over r to the 12th term and 1
over r to the 6th term.
First, notice that it's minus 1 over r to the 6 and minus
in potential terms means more stable in energy,
lower on this Y-axis of potential energy over here.
OK. So that's going to be our attractive term.
Hydrophobic, if things attract each other, OK, not just due
to repulsion from water.
We'll talk about that next.
But hydrophobic things want to stick to each other
and they're going to do this
with an attraction that's proportional
to 1 over r to the 6.
The fact that it's 1 over r to the 6 as opposed to r to the 2nd
in the charged-charged interactions means
that this is a much shorter range attraction.
This attraction takes place on a very tiny distance scale.
OK. Now eventually the two atoms in this case as described here,
or two molecules-- or two molecules bang into each other
and go past the point where they're attracted to each other.
OK? And at that point, their electrons are trying
to overlap with each other.
That's really bad news, right?
We know by the Pauli exclusion principle
that that's not allowed and so in the same way
that my fingers are never going to fuse with each other,
just going to bang off of each other, the atoms push away
from each other and they push away from each other
with the repulsion force
or repulsion potential that's proportional
to 1 over r to the 12th.
OK? And so this means that this is extremely short ranged
and extremely sharp, right?
To the 12th power is a large number.
So this means that this really dramatically pushes apart the
atoms if they happen to get too close to each other.
It turns out that there are a whole series
of other non-covalent interactions that we find
in biology that actually contribute quite a bit
of non-covalent binding energy.
Here for example are the dispersion interactions
that we have discussed before on the previous slide
and so these include things
like aliphatic-aliphatic interactions,
but also aliphatic interacting with hydrophilic molecules.
So here is water interacting with methane.
They're going to interact with each other
and have some attraction.
These number here, minus 0.5
to minus 0.7 kcals per mole is pretty low.
OK. This is not a tremendously strong interaction.
Where it gets strong is when you have a molecule
that has a large number of functional groups.
Each one with 0.5 kcals per mole here, 0.5 here, 0.5 here,
and when you sum up across all of these, you're starting
to talk about big energy.
OK. Now just to give you an idea, you need to know one fact
that I think is really important.
And the fact is important enough that I'm going to try
to write it on the board over here in the corner.
The fact is that, a factor
of 1.4 kcals per mole will be a factor of 10 in--
at equilibrium constants.
OK. So, 1.4 kcals per mole is a magic number
in chemical biology.
OK. So look for 1.4 kcals per mole
because that tells you then that's favored tenfold
over nonbinding.
In other words, the interaction is going
to be 10 times more likely to form than not form.
OK? It's a factor of 10 in terms of equilibrium constants.
OK. So, if we're talking about something over here,
that's only 0.5, 0.7 kcals per mole, you have to start summing
up a whole bunch of these to get anywhere in terms
of enforcing the interaction.
On the other hand, some of these other interactions can be quite
strong and let's take a closer look at those next.
OK. So, for example, we've talked a little bit
about hydrogen bonding.
Hydrogen bonding of course has a donor and acceptor
and here's a range of strengths.
Hydrogen bonds vary enormously strength
from about 1 kcal per mole all the way to 7 kcals per mole.
The strength of the interaction depends enormously
on the identity of the donor and acceptor.
When the donor or-- and/or acceptors are charged,
if either one is a charged functionality, the strength
of this hydrogen bond goes up enormously.
And this kind of makes sense, right,
because remember earlier I described a hydrogen bond
as a kind of a special case of a charged-charged interaction
in which a hydrogen is being shared between two atoms.
OK? So, if one of these happens to be charged, that's going
to be a much stronger charged-charged interaction.
Speaking of charged-charged interaction,
salt bridges are the coulombic potential
that we saw on Thursday.
These are the charged-charged interactions.
These vary also enormously depending upon the environment
that the salt bridge happens to find itself in, where a--
where water can shield this charge.
Water or counter ions can shield this charge,
decreasing it considerably
and making the interaction much, much weaker.
So, a salt bridging interaction, which is another way
of saying charged-charged interaction found
in a hydrophobic environment, say the interior
of a plasma membrane, is going
to be a much stronger interaction
than one that's found out in water where there's plenty
of water and counter ions to shield the charge.
OK, where those provide a counter against the charge.
Recall that those environmental terms are embodied by the 1
over 4pi epsilon term in the coulombic potential
that I showed you on Thursday.
OK. In addition, there's also dipole-dipole interactions
which are alignments of densities of charge
where we have a little bit more negative charge
on the oxygen over here.
The dipole is pointing in this way on the--
to the right on the upper acetone
and to the left on the lower acetone.
The two of these dipoles want to cancel each other out.
By cancelling each other out,
that will give you a more optimal interaction and that's
where some potential energy.
Finally, there's also a whole series of aromatic
or arene interactions.
And in general, this includes both face-to-face interactions
where you have two faces of a benzene ring
that are interacting with each other.
Notice in this picture over here
that the top benzene ring is offset
from the bottom one and this makes sense.
We're going to be looking at regions
of electron density interacting
with regions of electron poverty.
OK, that that's actually the basis for the interaction.
And so for that reason, we also see very commonly edge
to face interactions.
OK, so this is the one that we'll see in a moment
when we start looking at pi stacking in DNA.
But in addition, you can have an edge
of an aromatic system interacting with the face
of another aromatic system down here, and that's as strong,
right, it has the equivalent strength.
Even though you expect, you know, face-face to be ideal,
that's actually not what we see when we start looking
at large numbers of aromatic interactions.
We see this H-- edge to face interactions all of the time.
OK. And then finally there's some other ones
that are really bizarre and they include charged interacting
with the electron rich aromatic rings.
And this kind of makes sense, right?
You have something that's positively charged,
you have something that's very electron rich
in terms of the ring system.
So these cation-pi interactions
which is what this one is called are found pretty ubiquitously
in biology, oftentimes playing a commanding role,
playing a really key role in chemical biology.
OK. So, these are ones that I'd like you to memorize.
I'd like you to know something about their strengths,
which one is strong, which one is weak.
I don't want you to memorize the numbers per se but I want you
to know something and be conversant
on relative strengths.
OK, relative strengths matters.
OK. And one thing-- one last thing to keep in mind
if we're going for this 1.4 kcal per mole, again,
you can have a summation of a large number of interactions
to achieve that 1.4 kcals per mole or even more.
And I'll show you an example of that very shortly.
Now it turns out that it's actually a little bit tricky
to start comparing energetics when you design
in say the perfect cation-pi interaction.
What ends up happening is
that you get a complication due to water, OK?
So let's imagine that you had designed
in the perfect cation-pi interaction and in doing
so you put this positively charged thing that forces all
of the water around it
to rearrange itself or reorient itself.
It turns out that's actually a complicated thing
of the orient-- reorientation of water
but it cannot be neglected.
OK. So what we do is we make a very important simplifying
assumption, and I'll talk more about water on the next slide.
But before I do, water, since we just have to acknowledge
in advance, water can complicate everything, right?
It's present at 55 molar concentration in your cells
and we can't neglect it, OK.
It has its own energetics.
It's-- as I showed on this slide over here,
for example it's interacting with hydrophobic things.
So its own energetics are really complicated, OK,
and actually very hard for us to understand and pin down.
And so it's really difficult to estimate the entropy lost
or gained in an interaction due to that rearrangement of water
when you start making changes.
So what we'd like to do is compare things that are
as similar to each other as possible.
OK. This is the simplifying assumption
that I alluded to earlier.
Here for example is an example of that, OK?
So, here's two possible transitions states,
and transitions are two possible mechanisms.
Mechanism number one involves an SN2 reaction.
Mechanism number two involves the same molecule undergoing an
E2 elimination reaction.
And the key here is that the molecules are identical.
OK? That extreme similarity makes the comparison
between these two much easier to make.
OK. And so, for example if we're looking at two proteins,
we can look at empty protein versus ligand-bound protein.
But on the other hand, we're not trying to make all kinds
of changes to the protein structure over here.
Problem is proteins are rarely, you know, like looking like this
when the ligand is unbound.
So, these simplifying assumptions will start
to cause all kinds of problems.
Here's one though that works.
You can make a single change to the surface of a protein
and then compare the altered protein,
compare its interaction with a ligand.
So for example, we could change this isopropyl group
to a methyl group and then compare what's happened,
what's different in that receptor ligand interaction, OK?
So all you've done is to remove two methyl groups.
That's about as simple as it gets, right?
So that type of experiment is an easy one to make comparisons to.
OK. And again, by doing that, we're trying
to minimize how much the water has
to rearrange itself at that interface.
OK. It turns out actually this assumption works most
of the time.
So in short, being good scientist, not changing lots
of variables at the same time pays off in biology
because underlying everything we do is this complicated solvent
that we operate in called water.
Let's take a closer look at the structure of water.
OK, so here is water in ice and notice how neatly regular it is
and how nicely ordered it is.
And then here's water in a solution as liquid water.
And it's just crazy complicated.
First, notice that there, all these dots--
dotted lines are the hydrogen bonds.
These hydrogen bonds are pretty much maximized.
Water is not passing up any opportunities
to hydrogen and bond to itself.
OK, but the hydrogen bonds
in the liquid solution are nonoptimal.
OK, water in solution, each water molecule is jam-packed
with other water molecules
and oftentimes the hydrogen bonds are slightly distorted
or they don't have the right distances.
Those little distortions and that lack
of perfect distances makes the hydrogen bonds
in liquid water weaker than they are in solid water.
Furthermore, a molecule of water in its own, you know,
with a lot of other molecules of water is--
and behaving kind of like it's on a crowded dance floor, OK?
So, it's bouncing around wildly against these other, you know,
molecules that are nearby and interacting with lots and lots
of different molecules nearby,
constantly breaking interactions and forming new ones.
OK. So, water is actually very complex.
Weak and distorted hydrogen bonds, OK.
In addition, when water cozies up to hydrophobic surfaces,
it tends to form a very ordered structure that starts
to look a lot like the structure found in ice.
And this works by water form--
satisfies its propensity formed hydrogen bonds
by forming a clathrate-like structure.
So for example, here is a molecule of methane encapsulated
in one of these clathrates of water,
where clathrate is just simply a structure of water
that satisfies its desire to form hydrogen bonds with itself.
OK, or with other molecules of like kind, OK.
This really dramatically changes the strengths
of nearby non-covalent interactions, OK.
This does things to strengthen those non-covalent interactions
because every time one of those,
let's just say hydrophobic-hydrophobic
interactions breaks, then water has to slide
in between the now broken interaction
and form one of these clathrates.
OK, that formation of the clathrate, the formation
of an ordered structure cost energy.
It's a loss of entropy.
This is a more ordered structure than the structure
of disorganized water
that I showed you earlier in solution, OK?
So for this reason, hydrophobic--
hydrophobic molecules are driven against each other.
They want to find each other in water.
And this is sometimes referred to as a-- this is--
this is actually a water driven effect.
Forgetting the technical word for this.
OK, anyway, so-- oh sorry, it's--
it's sometimes referred to as a hydrophobic effect,
OK, in water.
OK, now let's take a closer look
at a receptor-ligand interaction,
now zooming in at the level of atoms and bonds.
This is a molecule called human growth hormone and, yes,
Lance Armstrong admitted to Oprah
that he took human growth hormone to win--
to help him recover basically from different stages
of the Tour De France during all seven of his victories,
and it really annoys me actually.
I could say a lot more about that
but I'm going to hold myself back.
OK, now when human growth hormone binds to its receptor
on the surface of cells, it's stimulating growth and recovery
of those cells, it's stimulating protein production, et cetera.
And when it binds to the surface of the cell
to the binding partner on the surface of the cell,
its receptor, then all of the region that's colored
in on this surface is buried, OK?
So in other words, human growth hormone binding protein binds
over here and then makes contact with each
of these colored atoms.
OK, everything that's in white here is still out in the water,
out in the solvent, it's not interacting
with the receptor at all.
Now, when I was at postdoc, I repeated a classic experiment
that was done by Jim Wells.
And Jim Wells and his co-workers found
that even though there are 19 residues that are buried
on the surface, there are 19 amino acids that are buried,
only the ones in red are actually contributing binding
energy, OK?
So, notice that all of these other stuff is in--
that is in blue that is buried is not
at all contributing any binding interaction.
So although there's interactions between these side chains
of these two proteins, there is no binding energy that's being
exchanged or gained by that interaction.
OK? So, just because two molecules find each other,
two functional groups find each other in space, does not ensure
that there's actually going to be a net gain in binding energy.
Because again, that net gain
in binding energy includes both the strength of interaction
but must also include the water ordering and disordering term
which we've been calling entropy earlier, OK?
So, in order for this interaction to take place,
you're going to be pushing out ordered water
and gaining some entropy in some places
and in other places losing some entropy.
OK, now when we look even more closely, let's just zoom
in on this red patch over here.
This red patch has been termed the hot spot of binding energy.
That's where the binding energy allowing these two molecules
to interact with each other is found.
OK, this is the essence of the non-covalent interaction
between human growth hormone and its binding partner,
human growth hormone binding protein.
And in green, these are the functional groups
that are found in this red patch.
OK? So the red patch is over here and now I'm showing you
that the functional groups were in green.
These are carbon atoms.
In blue, that's a nitrogen.
And in red, that's an oxygen.
OK, notice that the hydrophilic functionalities, the guanine--
of an arginine over here, a bunch of nitrogens,
another nitrogen over here, an oxygen, an oxygen over here.
Notice that those are around the periphery of this red region.
They're around the outside of this hotspot of binding energy.
The center of the hot spot is largely hydrophobic, OK?
Notice that it has lots and lots of carbons.
There's a benzene ring, it is smack in the center.
There's this aliphatic chain that's capped
by an amine functionality,
but nevertheless this is an aliphatic chain.
There's aliphatic functionalities over here
and over here and over here, et cetera.
OK, so in other words, the outside hydrophilic,
the inside hydrophobic.
And so, when molecules,
functional molecules find each other, this is a very common way
for them to interact with each other through a small set
of residues that form this hot spot of binding energy
which again kind of looks like a core sample through a protein.
Outside is hydrophilic, inside hydrophobic, OK?
Any questions so far?
OK, let's talk one last--
about one last section
of chapter 2 before we move on to chapter 3.
There's this concept that the biooligomers
on earth are highly modular.
We've discussed this before.
This also extends to the polyketides and the terpenes
which are composed of isoprenes and the polyketides
which are caused-- which are composed of either malonyl
or acetyl subunits that are strung together,
where the red bonds indicate that where the connection
between these modules such as the amino acids
as individual modules in a protein.
OK? And furthermore, this is also found in oligosaccharides
where you have this glycosidic bond
that connects the glycan fragments together.
There's also a numerical amplification and biosynthesis.
So, if there's only one or two copies
of DNA per cell depending upon whether it's a prokaryotic cell
or eukaryotic cell,
some prokaryotic cells are [inaudible] more than one
but let's just simplify it.
Then to RNA, each DNA is transcribed 10 to 50 times
and then each RNA is translated say 10 to 20 times.
So in the end you end up with this massive amplification
signal going through the cell, where with one copy
of DNA you can end up with millions of products
from some enzyme reaction down here.
Last thoughts, form follows function in biology.
These-- the bonds that join together,
the oligomeric subunits are--
have a strength that follows their function,
their functional requirements, OK.
And so, for example, when we look at the half-life of lipids,
we find that actually the ester bonds in a lipid have a halfway
on the order of a year or so.
OK, so esters, not so stable.
Compare that against DNA down here which has a half life
on the order of 220 million years.
OK, that's his half life for DNA.
And in retrospect, this kind of makes sense, right,
because DNA has to be a-- you know, has to be a biooligomer
for the life of the organism, OK?
And so, we're now at the point
where we're routinely taking advantage
of this tremendous stability of DNA to amplify DNA
from even extinct organisms like wooly mammoths, like species
of prototypical humans that haven't lived on the planet
for tens of thousands of years.
That sort of thing is going on right now
in laboratories talking advantage
of the tremendous stability of DNA.
Now your hair, which is a protein, has a lifetime
on the order of, you know, 300 years or so.
And you can see that, right?
We can find, you know-- we give-- well, anyway.
So, I guess it depends on the human that we're talking about.
My hair obviously doesn't exist that long.
But, you know, so certainly the lifetimes here are following
their function, right?
Proteins don't have to last this long.
Question? How does one get a PhD that's going to take you five
or six years studying and trying
to measure these half lives of 220 million years?
Anyone have any ideas how to do that experiment?
I can guarantee it to you, it's not like, you know,
you set up this test tube
and then you check it every 20 years, OK,
to see how much gets cleaved.
How would you do this?
Yeah, how would you do it?
[ Inaudible Response ]
OK, a small amount of RNA.
And I would use a large amount
because very little is going to get degraded.
How would you do this though?
Yeah?
>> Maybe we can put it in a very decomposing environment?
>> OK. But then you wouldn't know
if the decomposing environment is different
than in the cell, right?
We want to know about the half lives in the cell, right?
Yeah?
>> Use a model organism.
>> Model organism.
No, I want to know what it's going to be--
what's going to last in, you know, in this cell
or this other one over here.
Question over here?
[ Inaudible Question ]
OK, you're definitely going to use radioactivity
because you need something that's super sensitive.
How would you do this?
[ Inaudible Response ]
OK, you're getting close.
What is your name?
>> Bryan.
>> Bryan? OK, Bryan is getting close.
So the suggestion was radioactivity.
Bryan's suggestion is you look for a tiny little quantity
and radioactivity gives you that sensitivity.
But are you going to do this
for 220,000 years or 220 million years?
>> No.
>> OK, so how are we going to do this experiment?
We have the sensitivity, we're going to look
for tiny little quantities and extrapolate back.
How are you going to model 220,000 years?
Yeah?
[ Inaudible Response ]
Carl, OK, look at fossils.
Yeah, and we do that.
Yeah?
[ Inaudible Response ]
OK C14.
[ Inaudible Response ]
OK.
Yes.
>> You would compare it to that.
You note the reactivity of one and then you compare it
to the one that you know.
>> Ahuh.
>> And it is-- and extrapolate that half life,
half life, half, half, half.
>> OK, so-- but the problem is you wouldn't know all the
conditions that's experienced over, you know,
say 100,000 years or something, right?
So, I mean, how do you--
you want to do this in a controlled circumstance.
You want to have everything just in a little test tube
where you know exactly what's been added
to the test tube, right?
But you don't want to wait around for 220,000 years
or 20 million years, what are you going to do?
OK, I'd like you to look this one up.
This is one that you should be able to design.
Look it up.
And then when we come back on Thursday we'll talk about this.
But I'd like everyone to have to look this up, OK?
This is important.
OK, let's talk--
let's summarized what we've been talking about in terms
of non-covalent interactions.
These are completely ubiquitous in biology.
Good news, we only have to learn two equations
which govern all interactions in chemical biology.
Those were the Coulomb's law for the charged-charged interactions
and the Lennard-Jones potential
for the uncharged interactions, OK?
And so if we know those two equations, we're set.
What's really-- and what's important to us is not
that we're going to be plugging in, you know, charge of this
and then, you know, radius of this.
What's important to us are the relationships,
the distance dependence,
the 1 over r squared versus 1 over r 6.
That type of distance dependence makes a big difference.
And knowing that sort of thing and having sort
of an intuitive grasp of that is going to be very important.
So-- and I'll just give you a quick example.
For example, we now know if DNA is negatively charged,
it's going to attract other charged ions to it
from great distances, right?
Because it's distance dependent, it's only 1 over r
to the 2nd power versus1 over R to the 6.
In addition, we've learned
that these non-covalent interactions are very sensitive
to the environment, the distance and the geometry.
Water is a really slippery molecule
to understand, to say the least.
Has a malleable structure
and it can dramatically alter the strength
of non-covalent interactions.
This makes it really tough for us to draw any generalities
because water is an intermediate lubricant between all
of these interactions and it plays a complicated
and sometimes hard to us-- it's hard for us to define role.
And there's still big arguments that are going
on in water chemistry to this day.
For example, there's an argument going
on about how many ions are found on the surface of water
or what's the pH at the surface of water.
And there's been a set of dueling papers
that have appeared that contradict each other.
The first paper had a title like the pH of the surface
of water is more acidic.
The next article-- the next article
by the competitor said the pH of water
at the surface is more basic.
And the two-- and these groups have been arguing backward
and forth and both making very reasonable arguments for years.
OK? The truth is what we found is actually it's somewhere
in between these two and you can actually see evidence
for either one and it turns
out to be a very minor effect that's not
so important in biology.
But the point is is that water itself is
such a complicated fluid
that we're still using the latest techniques to try
to understand it better.
It's not fully understood.
Hydrogen bonds have donors and acceptors
and they are also very susceptible to a competition
with water for those hydrogen bonds.
I would like you to know the approximate strengths,
the relative strengths, not the approximate
but the relative strengths, and distance dependence
of non-covalent interactions.
That's important.
OK, so that's a summary of chapter 2.
Any questions about chapter 2?
Yes, Chelsea.
[ Inaudible Question ]
Yeah, I really want you to know that.
OK, that's super important.
That's that Henderson-Hasselbalch equation.
That hopefully you learned in Chem 1,
you definitely need to know that.
Other questions?
OK, let's move on.
I want to talk to you about the structure of DNA.
This is the classic structure of DNA first proposed by Watson
and Crick in I believe 1952 or-- yeah, 1952, somewhere in there.
The structure of DNA has two strands running
in opposite directions to each other.
So they're anti-parallel to each other.
The strands are held together by phosphodiester bonds
which we'll look at more closely.
So, here's a schematic diagram of what the structure
of DNA looks like and here's a space filling view
where each one of these spheres is a van der Waals' sphere
to approximate where the atoms are,
where the outermost electrons of the atoms are.
One thing to notice is that DNA has two grooves, OK?
It has, yeah, the distance here
between these two strands is very close
versus the distance here between the two strands being much
further away.
These are going to be called the minor
and major grooves respectively.
And this is the origin of the fact that DNA is a double helix.
I think it's commonly thought that DNA is a double helix
because it's two relatively rod-shaped molecules
that are twisted with each other.
But that's actually not the case.
It's a double helix because it has a minor groove
and a major groove.
And I believe the next slide will show us that more closely.
OK. So, in blue, this is the major groove of DNA,
and in green, this is the minor groove.
In red, this is the phosphodiester backbone of DNA
that we've seen before, OK?
So again, notice that there are two helices
that are running parallel to each other, a major groove
and a minor groove, OK?
The structure of the bases is going to set up this major
and minor groove relationship.
As we will see shortly, DNA bases, base pairs form a U shape
and that U shape ensures that you're going to get a major
and a minor groove, where the inside of the U is going
to be this minor groove and the outside will be the
major groove.
But I'm getting a little bit ahead of myself.
The reason why this is important is as we'll see in a moment,
proteins like to interact with the major groove of DNA,
whereas they can't fit in to the much closer interstices
of the minor groove of DNA.
Rather small molecules will fit into this minor groove and try
to largely avoid the less cozy major groove of DNA, OK.
So, almost immediately we can start to make some predictions
about where stuff binds just knowing
that DNA is a double helix, double by virtue of the fact
that it has two parallel helices,
minor and a major groove.
So, this DNA structure immediately sets up replication.
This is the original 1953 paper by Watson and Crick,
and this is the very last sentence of the paper
in which they had this incandescent understatement.
It has not escaped our attention,
it has not escaped our notice
that the specific pairing we postulated immediately suggest a
possible copying mechanism for the genetic material, OK?
So, if you have two strands of DNA running anti-parallel
to each other, you can simply separate out the two strands
and then get a perfect copy of one strand over here
and a perfect copy of the second strand over here, OK?
So, here's the parent strand of DNA and again,
here are the two new strands in orange and blue.
Note too that DNA forms a right-handed helix.
OK, does everyone see that you can trace out along the right--
with your right hand over here the structure of DNA?
I think it's worth trying that.
Whereas your left hand kind of slips off,
it doesn't trace it out effectively, OK?
Does everyone see that?
So, it's DNA is always a right handed helix.
You know, so this beautiful structure of DNA is one
that was solved by x-ray crystal structure.
Before then, there were a large number of wrong,
incorrect predictions about DNA structure, including by people
who I, you know, think the world of, I think are, you know,
absolute heroes in science.
For example, the great Linus Pauling
who proposed a triple helix of DNA
where the phosphodiester backbones would be in the center
of the molecule and the bases would be out on the outside.
This kind-- this is somewhat--
this is intellectually attractive if you don't think
about the fact that you have two parents.
But furthermore it's attractive
because at least the base pairs would be out here in space
where they can interact with transcription factors.
We now know of course that that's not correct.
Instead, we'll take a look in a moment
at where the transcription factors interact.
Before we do, let's zoom out a little bit, OK?
So, DNA in the cell is concentrated in two regions,
a nucleosome in the prokaryotic cell, so it's kind
of concentrated in the very center of an E. coli cell.
In a eukaryotic cell of course, DNA is found exclusive--
is found in the nucleus and also the mitochondria
but let's just focus on DNA that's in the nucleus for today.
The bases themselves are connected together
to form oligonucleotides through this phosphate,
this phosphodiester functionalities, OK?
So this is called a phosphodiester functionality.
The DNA also has a directionality associated
with it, OK.
So there are-- if we look closely
at this deoxyribose base, there is a 5 prime end,
there's a 5 prime hydroxy over here
and a 3 prime hydroxy over here.
And so, the convention is to always write DNA
in the direction from 5 prime to 3 prime.
In the same way that we read English going left to right,
DNA is always read out 5 prime to 3 prime.
This is a really important convention, OK.
Everyone on the planet follows this convention and I'm going
to hold you to it as well, OK, because if you read the DNA
in the opposite direction you get a different--
or different word coming out, OK.
It spells something else that might not be this--
it will almost certainly not be the same thing
and it might actually be, you know,
might actually cause a lot of trouble.
So we're always going to be reading this 5 prime
to 3 prime directionality, so this sequence here would be read
out as A, C, G and T, OK, where the structures of A, C,
G and T are shown here, OK.
Don't bother memorizing this-- sorry.
Don't bother memorizing the structures of these.
I'll simply give them to you on the midterm, OK?
So, at a graduate level, you should know this.
Mariam will need to know this for her orals exam, but the rest
of you are in luck because I'm not going to test you on them
at least for this class, OK.
And again, the-- the directionality matters a lot.
If there is a 5 prime phosphate,
this 5 prime phosphate is indicated
by a lower case p. Finally, last bit of nomenclature,
oligonucleotides that are connected together are often
referred to as oligos and that's how I'll describe them.
OK, now I realize oligos is not the most descriptive
nomenclature because it just simply means an oligomer
or something, but that's the convention
that we've been operating under 50 years, OK.
So oligos will refer to oligonucleotides.
Typically DNA oligonucleotides compose of deoxynucleic acid.
OK. Now, even though DNA is--
the bases of DNA are called bases, it turns out they're not
that basic and few are protonated
at physiological pH. It's--
this is kind of one of those historical anomalies.
Here's a bunch of pKa's,
for example starting with triethylamine.
Here is the pKa of the protonated triethylamine,
the conjugate acid of triethylamine, pKa of 10.8.
Here is the pKa of cytosine, thymine, adenine and guanine
and you can see none of these would be remotely
considered bases.
Whereas this one over here, triethylamine definitely a base,
OK, as evidenced by the fact
that its conjugate acid is, you know, 10.8 pKa.
OK. Question so far?
All right.
Now DNA of course is missing a 2 prime hydroxyl, OK.
So here is RNA, it has a 2 prime hydroxyl over here.
This 2 prime hydroxyl makes RNA considerably less stable
than DNA.
I didn't point this out-- let me go back to it--
when we talked earlier about half lives.
Let me just zoom back to that really fast.
The half life of RNA is considerably lower
than the half life of DNA, OK.
So, here is the half life of RNA, 220,000 years,
whereas the half life of DNA
at 220 million years is much, much greater.
OK, a thousand fold difference in stability
for the phosphodiester backbone of the DNA
versus the phosphodiester backbone of RNA.
This makes sense, OK?
The 2 prime hydroxyl of RNA sets you
up for hydrolysis using an intramolecular attack, OK?
So, here's again the structure of RNA.
Here's the 2 prime hydroxyl.
This 2 prime hydroxyl can act as a nucleophile
to attack the phosphodiester backbone
of the RNA setting up cleavage.
Does anyone want to see the mechanism of that?
OK, all right, let's take a quick look.
OK, so in this mechanism--
[ Pause ]
Let me just draw up the structures
and then I'll blank the board.
OK, one second.
OK. So, in this mechanism, here's our structure of--
[ Pause ]
OK, so here is our backbone structure of RNA
and I'm just going to draw this as base over here, OK?
OK, so if there is any base that's present,
let's just say hydroxide,
this can deprotonate the 2 prime hydroxyl, giving us an alkoxide.
[ Pause ]
-- adjacent
to the phosphodiester backbone of the DNA.
This neighboring alkoxide can now attack the backbone,
the phosphodiester backbone,
giving you a five-membered ring intermediate.
OK, which I'll show down here.
[ Pause ]
Five-membered ring intermediate
and this intermediate collapse leading to cleavage of the RNA.
OK. So here is that collapse.
OK. So, we're going to be making two strands of RNA
that are separated from each other.
[ Pause ]
OK, so here's one strand over here
and then here's the second strand down here.
[ Pause ]
OK. I'm going to just differentiate this
as base 1 and base 2.
OK. So, notice that the strand has actually cleaved apart.
You can then hydrolyze this phosphodiester backbone,
this phosphodiester back
to a phosphomonoester using another equivalent of hydroxide.
[ Pause ]
And then finally, collapse
of this tetrahedral intermediate gives us the product.
OK. Questions about this mechanism?
All right, now notice again, if DNA lacks this 2 prime hydroxyl
over here, and I just want to make this totally explicit,
I'm going to label it 2 prime hydroxyl, 3 prime, 5 prime.
OK, so DNA lacks the 2 prime hydroxyl
and therefore does not have an opportunity
for this intramolecular nucleophilic attack
on the phosphodiester backbone.
So, for this reason, DNA is a thousand times more stable
than RNA, right, lacking this intramolecular nucleophile.
Makes sense?
Questions about this?
OK, let's go back.
Turns out that when you look at the liability of the bases,
we see actually a different trend.
OK. And actually I think I'm going to skip that.
OK, moving on.
OK, I'd like you to learn what I just told.
Don't worry so much about the base stability.
DNA bases are subject to important modifications.
These modifications have dramatic roles
on the phenotype of organisms, OK?
So, for example, methyl groups are often transferred to DNA.
I showed structures of DNA bases.
Again, they're subject to massive modification
by methyltransferases and other modifications.
So, for example, here's 5-methylcytosine over here,
4-methylcytosine and then N6-methyladenine.
These modifications can dramatically alter
transcription levels.
They can set up the organism
to transcribe some genes more often, OK.
So, for example, lacking pigmentation,
the genes that encode pigmentation are
in my skin cells, my epidermal cells,
yet they're not transcribed very often.
And so, it's likely that my DNA has not been methylated
in those regions.
However, when I go out and spend a lot of time in the sun,
I'm getting additional little spots called freckles
which are resulting from methylation
of those DNA sequences which in turn then turns on transcription
of the pigmentation and results in freckles, OK?
So, the environment, the environment that you're exposed
to can alter these transcription patterns.
It's one of the ways that organisms like ourselves respond
to changes in the environment.
It's a very important way in fact.
And oftentimes this goes through methylation of DNA.
This DNA methylation is really as important as sequence
or genomics, and this is an area called epigenetics.
That's really an area of very active research that's taking
place in chemical biology.
OK. So, we've looked at structures
of the bases themselves, we've looked at structures
of the phosphodiester backbone,
let's start putting things together to start
to understand the structure of DNA.
The bases themselves are slightly U-shaped, OK.
So, here's a base between A and T, adenine and thymine.
Notice that this base is composed of two hydrogen bonds.
Here's a base of G and C which has three hydrogen bonds.
But notice more importantly that the bases are U-shaped
or equally importantly, OK, U-shaped here.
The inside of this U where the R is going
to be towards the ribose, the deoxyribose ring,
the inside of this U is going to form the minor groove
which I've showed you on an earlier slide.
The outside of the curvy part of this U is going
to form the major groove.
As you have these U's that are stacked on top of each other
and each one is slightly offset with each other, this is--
outside is going to result in a much bigger helix
than the inside over here, OK?
And here's what this looks like.
OK. So, here's a trace of the phosphodiester backbone
and then I've highlighted just one Watson-Crick base pair, OK.
And again, notice that it's U-shaped,
that there's more section traced out over on this side,
that will be the major groove,
and the inside will be the minor groove.
Furthermore, the green arrows define hydrogen bond donation
and acceptance by the base pair.
And notice that there is a pattern to this,
that there is an acceptor-acceptor donor, OK.
So, this is a donor acceptor donor over here.
So, there's actually a little bit of a pattern
to whether this is a G on this side and a C on this side or C
and G on the opposite sides.
So, in other words, A and T are not the same as T and A
because they're going to present a different pattern
of hydrogen bonds for molecular recognition where again,
the proteins are going to be--
the transcription factors are going to be interacting
over here in the major groove
and small molecules would be interacting
in this minor groove down here.
I should mention that there's also some protein DNA
interaction in the minor groove.
It tends to be more minor, however.
OK, let's take a close look at one example
of a transcription factor and how it works.
This is the transcription factor, Fos-Jun,
it consist of a leucine zipper which is two helices
that interact with the DNA like chopsticks, OK.
So, these are fitting neatly in the major groove.
It turns out the major groove has exactly the right size
to accommodate an alpha helical protein, OK.
So, this Fos-Jun is absolutely perfect.
It fits neatly in the major groove.
Now, these hydrogen bond donating functionalities are
going to then read out the sequence of the DNA and look
for a specific sequence of DNA to interact with,
trying to form complementary hydrogen bonds,
trying to form complementary van der Waals interactions
in this sequence, OK?
Let's take a closer look now
at the forces holding together the DNA double helix.
Earlier, I alluded to the fact
that AT base pairs form two hydrogen bonds
and GC base pairs form three.
Which one is stronger, just, you know,
from a crude approximation?
Yeah, three is stronger than two, right?
OK, so in addition to this, the DNA structure is held together
by pi stacking between the bases.
Again, this is a face-to-face interaction.
Typically not perfectly face-to-face,
rather it's typically offset.
And that offset needs the bases to stack not directly
on each other but slightly twisted from each other,
setting up this helical structure
that we're now familiar with.
In order for this base pairing to take place, the base pairing
that I showed on the previous slide,
you need a particular tautomer of these aromatic rings, OK.
And the first one that should strike you as funny is this one
over here, because you can imagine another resonant
structure that would make this C aromatic, right?
Notice that the C has--
is non-aromatic in this tautomer shown here, right.
It only has two pi electrons rather than the requisite six
that it would need to be aromatic.
OK, that's almost-- that's bizarre to begin with, OK?
So, what's going on here is that there is a preference
for this tautomer versus this one.
This one is actually thermodynamically more stable
and the reason for this is that the carbon-oxygen double bond
over here is quite strong.
I will tell you that I think any chemist looking
at this could not have predicted this in advance,
and in fact actually this tremendously slowed structure
determination of the original structure
of DNA back in the 1950s.
Watson and Crick were physicists and weren't as familiar
with the whole notion of tautomerization
as their chemical counterparts were racing
to solve the structure of DNA.
And so for them, this did not look funny whereas
to us I think it does look funny, right,
because it lacks aromaticity whereas a structure
on the left is aromatic.
Again, this happens to be just a little bit more stable
because of the strength of the carbon-oxygen double bond,
but I don't think anyone would have predicted that, OK?
I think now we, you know, with our 21st century guys,
we could predict it, but going back in time,
I don't think we could have predicted it so readily.
Similarly, over here, these amidines are actually going
to be more stable in the aromatic structure
than an amidine structure.
And in this case, that's due to the much poor overlap
between a carbon-nitrogen double bond
than a carbon-oxygen double bond, OK.
So, all of these lead to the base pairs
with the hydrogen bonding preferences
that are shown here, OK?
Whereas for example, this is a non-aromatic ring
that could aromatic if it tautomerize,
but it doesn't prefer to be tautomerized.
Whereas this one over here seems to prefer to have an amidine
in this structure because of the strength
of a carbon-nitrogen double bond, OK?
And here is another example of that over here.
This one prefers aromatic
because carbon-nitrogen double bonds are relatively weak.
OK, pretty interesting.
Unnatural bases, however,
could dramatically shift these preferences for tautomerization,
and a good example of this is 5-bromouracil, OK.
So, if this compound here is fed to organisms,
what happens is an unusual tautomerization preference
where the enol form of bromo-U is actually more preferred
than it would be if there was no bromine over here, OK?
So, most of the time, it forms the regular base pair, however,
some of the time,
it can actually form the incorrect base pair
because it can actually more readily access this enol form
of the base, OK?
So, that's due to the electron withdrawing functionality
of bromine over here, OK?
That's changing this tautomerization preference.
The consequences of these are really dramatic.
Because the Watson-Crick base paring is not followed
as closely, what ends up happening is the DNA comes
out with all kinds of bizarre breaks and lesions, OK.
So here are chromosomes from a normal organism.
I think it's a hamster in this case.
And then here's chromosomes from hamsters that were exposed
to bromouracil and you could see they have all kinds
of bizarre shapes to them, things are incorrect, OK.
So, this causes cancer and breakages in DNA
which then eventually lead to cancer, cancer cells
and tumors in the organism, OK.
All right, so furthermore,
it turns out that we can test this--
the importance of the strengths of these hydrogen bonds
by synthesizing unnatural bases.
So, this is one of the great things about chemical biology.
If you have this hypothesis that something is important,
then you could test that hypothesis
by synthesizing compounds which are say missing
that key functionality.
So, from Watson and Crick, we expect to find
that hydrogen bonds are holding together the structure of DNA
and chemists went out and synthesized variants
of DNA bases that were lacking
that ability to hydrogen bond, OK?
Structures of these are shown here, OK?
So, for example, this compound here is simply a pyrene in place
of a base and it actually prefers to base pair
with a missing base over here, OK?
So, these guys over here, no hydrogen bonding,
no hydrogen bonding over here and yet these actually prefer
to pair with each other, OK.
So, you can actually have completely unnatural bases
missing hydrogen bonds that is-- are yet able to form base pairs
with each other preferentially.
What this tells us is that there's more going
on in DNA structure than simply hydrogen bonding.
Hydrogen bonding is a nice simplifying assumption
for our biochemical friends or molecular biology friends,
but in actuality, the pi stacking
of DNA is a driving interaction, the edge to edge interactions
of aromatic functionalities are also driving these interactions
between the strands of DNA.
And so, while we can do quite a bit with hydrogen bonding,
there's quite a bit more that's left to be explored.
OK, last thought, I've been showing you--
or it's not last thought, I've been showing you--
Oh, before I get to that, here's-- here for example is--
this illustration here emphasizes the importance
of pi stacking in here, OK?
So, one thing is that bigger bases tend to pi stack better,
for example, the guanine base tends
to pi stack better than say cytosine.
All right, in addition,
I've been showing you Watson-Crick base pairing
where it's a canonical base pair,
G's and C's have three hydrogen bonds,
A's and T's have only two.
Other kinds of hydrogen bonding possibilities are not only
possible but have been observed.
These were proposed by Karst Hoogsteen
and we observe this a lot in RNA structure.
We don't necessarily see this in DNA, but we definitely see this
in RNA and they're going to come up later.
So, I'll just show you the structures here.
This is an alternative to the usual AT base pair
and this is an alternative to the usual CG base pair.
This one being driven by a protonation event,
protonation of this nitrogen over here, OK?
So, this is actually-- these are sort of edge
to edge interactions rather than the sort
of neat more typical Watson-Crick base pair.
OK. Any questions about the structure of DNA?
Anything, whatsoever?
I want to change gears then and start talking
about how small molecules interact with DNA.
The first mode that small molecules can interact
with DNA is to actually slip into this pi stack of DNA.
So, aromatic compounds can slide into the pi stack of the DNA
and we're going to see the consequences
of this can be quite destructive.
Let's take a look at some examples, this is a class
of molecules called intercalators,
meaning that they intercalate into the pi stack of the DNA,
they get integrated into the DNA structure.
So, in order to fit into this pi stack,
these molecules must be also hydrophobic
and also aromatic, right?
They will form competing pi-pi stacking interactions
with the DNA and so they must also be aromatic.
Note too that in order to force the way into the pi stack,
these molecules force the DNA's double helix to slightly unwind
to accommodate the DNA intercalator.
Here are some examples of this.
These are examples of intercalators.
Notice that they are all aromatic compounds.
They're all flat and aromatic to slide into the pi stack.
Many of these molecules also have positive charge.
Positive charge is useful, right,
because DNA with the phosphodiester backbone
of the DNA is negatively charged.
This gives the molecule a way to be attracted in the DNA
through a long range charged-charged interaction,
right?
So these molecules are going to seek out DNA
like a homing missile.
And once they slide into the pi stack,
the consequences can be pretty bad
or actually fairly useful, OK?
Let me show you an example of a useful intercalation
over here on the right.
This is actually an agarose gel which is an important way
that chemical biology laboratories separate
out DNA structures.
Different DNA sequences can be separated out on the basis
of their size using these agarose gels.
I'll show you what that looks
like in a couple of slides from now.
To visualize the DNA, however, this molecule over here,
ethidium bromide is incorporated to the gel
and it gets concentrated into the DNA
by an intercalation interaction.
So, it slips into the pi stack of the DNA
and it's a fluorescent molecule,
many aromatic compounds are fluorescent.
We've talked about fluorescence before.
And so, you can actually shine UV light on the gel
and wherever you see this-- these pinkish bands,
that's where the DNA is present.
And so you can actually take a razorblade for example and cut
out the DNA of a particular size.
Here's a couple of more DNA intercalators.
Here's one that's designed to intercalate
and then have a little linker and then intercalate
down below the compound.
Here's what it looks like structurally,
so there's intercalator, linker,
intercalator up here for example.
I think that-- this it right over here.
These are also compounds that are used to treat cancer.
So, dynemicin, adriamycin are used as anticancer compounds
or some of the first rounds of anticancer compounds
that are used as chemotherapeutics.
And we'll talk more about their mechanism
of action later in the class.
We're not quite there yet.
OK. Let's stop here.
When we come back next time, we'll be talking more
about the structure of DNA. ------------------------------0b468b059f88--