Lecture 06. DNA Reactivity with Small Molecules.


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Lecture 06. DNA Reactivity with Small Molecules.
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Lec 06. Introduction to Chemical Biology -- DNA Reactivity with Small Molecules
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Weiss, Gregory Alan
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University of California Irvine (UCI)
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UCI Chem 128 Introduction to Chemical Biology (Winter 2013) Instructor: Gregory Weiss, Ph.D. Description: Introduction to the basic principles of chemical biology: structures and reactivity; chemical mechanisms of enzyme catalysis; chemistry of signaling, biosynthesis, and metabolic pathways. Index of Topics: 0:01:53 Examples of DNA Intercalators 0:04:21 How to Measure and Calculate the Strength of DNA Hyrbidization 0:09:20 Short Stretches of DNA & RNA Can Also Fold 0:10:31 DNA is Wound into Supercoils 0:15:53 Bacterial DNA is Stored in Plasmids 0:18:29 Antibiotics as Selection Markers 0:22:24 Eukaryotic DNA is Wrapped Around Nucleosomes 0:27:24 Trapoxin as a Highly Specific HDCA-1 Inhibitor 0:28:49 Biological Polynucleotide Synthesis 0:32:07 DNA Polymerases 0:46:57 RT Inhibitors 0:52:15 A Brief HIstory of Chemical DNA Synthesis 0:57:01 The DNA Microarray 1:02:09 FK506 Fingerprinting 1:03:58 Analysis of DNA by Electrophoresis 1:07:55 DNA Sequencing Uses DNA Synthesis 1:10:37 DNA Biotechnology
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OK so hopefully I wasn't too painful I realize that might have been but if you put your name on a piece of paper you will get some points that I promise you and edition of the next time I see that there's something I'd like to go look up I hope for you to get worse you take it seriously ,comma but don't panic but hopefully not too painful and let me just tell you very quickly the answers OK so that last time we saw the half-life of DNA was on the order of 220 million years and I challenge you to go out and find out what it had a measure of and lease 1 person came to my office tower with a solution which is the the DNA seeking heated up and at a high temperature measured the half-life at a higher temperature and extrapolate back to the lower temperature and you don't have a variety of a lot of equations to show that that's true but if he does write heat on our problems and number 2 I will accept a but again if you have your name on a sheet of paper with 3 CID number and we'll give you get at least some points OK OK I'm sorry to say that we're back to normal stuff now I also want to pick up where we left off last time we were talking about the structure of DNA
and reactivity of DNA and we saw
last time produced here
1 moment OK we saw last time a lot about Watson Creek base pairing and structures of DNA In its media they form now 1 thing I got asked about immediately after the lecture was you know as
bromide is commonly considered to be a carcinogen this molecule over here that we discussed as an intercollegiate DNA is also a carcinogen it's a molecule that causes cancer and that's the reason why when we work with that in laboratory over here were exceptionally careful to keep it off of our skin he came down and yet it's interpolated to DNA and DNA interpolated can cause I can't help to initiate carcinogenesis scanning helped initiate cancer in the following ways number while it distorts the structure of DNA I showed you that on the previous
slide over here where I
showed you how the structure of the of the DNA has to unwind to accept and Terkel later so it distorts the structure of DNA number 2
it places a hydrophobic
functionality in the center of the DNA this hydrophobic functionality 10 in the appropriately tracked transcription factors to the DNA setting off it setting off incorrect transcription of so visit to possible moves that this can start to cause of statements can start to initiate improper cell responses that will eventually lead to cancer and I want to talk about answer today it's 1 of the prime topics of in this class it's something we're going to spend up quite a bit of time discussing in many different context and will certainly be talking about it in the context of the OK so any other questions about what we saw last time there was a really good question OK last thought about bromide although it is definitely if it is a cancer-causing agent and is something you definitely do not want to ingest a immediately want to keep it off her hands I don't want to exaggerate its carcinogenic potential but it's also used as an antibiotic and she had enough it still is but it was for a while but it does have some other actually spent a sheet of metal why she bites of in any case as a as a tumor for mediation and its activity is rather modest especially compared to the other cancer-causing small molecules that are going to see a very shortly had art that's a dive right in the I'm so
last time I was showing you that DNA likes the form Watson Crick base this is binding to G is a duties and we saw that she sees Watson Crick pairs have 300 bonds and 18 so we have to OK now if we know that that 18 base pairs are weaker than sees there's a very simple rule that we can use to estimate the strain of any 2 DNA strands sticking together this rule is called the Wallace rule and I'd like you to memorize affected the Wallace rule tells us that the approximate melting temperature for DNA us sequence is equal to 2 times the number of 18 base pairs plus 4 times the number of GC base pairs in degrees this melting temperature is where 50 per cent of the DNA is no longer forming a double-stranded structure let me explain so when you do you pay you believe it is honesty and of DNA and this is what here's what you fight a case of this observance online access and wavelength in 90 meters on the x-axis and this is single-stranded DNA and then the art double-stranded DNA in blue and and so going to get this you simply have higher temperature so at higher temperature the DNA strands Milt apart in other words they separated out into the 2 arms a single strands and so at 82 degrees this is the single stranded at 25 degrees this is the double-stranded DNA noticed that the absorbency at 269 meters is higher for the single-stranded DNA the double-stranded DNA so you can use that change in absorbency to follow whether or not you're DNA is single-stranded versus double-stranded and so you can do this while at the same time you ramp up the temperature and about the 50 EPA said so here it is pure double-stranded DNA lower observance and here it is at a higher temperature worked entirely single-stranded DNA and the approximate 50 said part with 50 cent is melted is called a melting temperature and again you can't estimate what this melting temperature is using this Wallace rule the formula people in chemical biology laboratories uses Wallace rule formula on a daily basis of certainly my laboratory and certainly in probably 5 or 6 other laboratories here survived so I'd like you memorize this rule it's incredibly useful now you're probably wondering big deal so I know whether or not something else whether or not it forms double-stranded DNA if you know and the other at temperatures that forms double-stranded DNA you can start design structures me out of DNA let me show you pay so
here are structures of DNA where its single strands of DNA that are now hybridizing against each other and forming elaborate patterns such as this pattern here and here's an atomic force microscope image of this double-stranded DNA could see it's all it's forming this exact cross-linked pattern that was designed using something just a little bit more complicated than the Wallace rule which I asks you to memorize pay it gets even better check this out
as this is work done by Paul Rofe inland and colleagues and coworkers at Caltech and on he's using the Wallace rule to design DNA that folds up into happy faces over here or on check out this map of the world written out of DNA that's been folded up with itself case that Wallace rule I ask you to memorize is actually pretty powerful you can develop whole structures the stuff now exactly what the structures of DNA are going to be useful for is not 100 per cent noted this point and there's sort of a frontier a chemical biology building structures out of DNA and trying to do something useful with them I've only seen 1 paper in 20 years of staring at these beautiful pictures that has convinced me that maybe there might be something useful about this and in that paper this DNA and DNA structure like this 1 not the happy face that something elaborate and was used as a delivery vehicle that down to the surface of cells and then dislodged a drug therapeutic and it's possible that our future might feature many more of the sort of examples of 9 steel structures that are designed by by you by the people in this room to I'm have specific properties such as binding to specific cell types unloading cargoes at specific times etc. This is a really exciting frontier and I urge you to think about it in your proposal preparation because it's an area that's kind of wide open for created for creativity OK
so I'm in addition to those sort of macro structures of DNA that we stopped short stretches of DNA can also called and there's a couple of canonical structures that were going to see time and again 1 of these for example of what is called here are called hairpins 1 of these is called the hairpin and so this consist of a sequence that holds back on itself that is that it satisfies all of the Watson Crick steering requirements GCC's faced tease and on something that looks kind of like an old-fashioned here .period and it looks structurally like this this is the X-ray crystal structure of what it looks like it again we're going to see this from quite a bit of a case of DNA has a propensity to Folden itself it of once a form secret base fears it wants a form Watson Crick base pairs with other sequences it wants the foreign base pairs with itself and so for this reason and DNA is rarely found a sort of it unwound which really ,comma single-stranded DNA for for 1 thing and furthermore it's rarely found in a sort of canonical B DNA confirmation that I've been showing you where it's it's nice right-handed quilt but a double helix rather than in cells we typically find DNA In in allowing the configuration called a super Quayle so a super Quayle is where you take a coil such this old-fashioned telephone cord which assures unfamiliar to everyone in this room look back invited kids we used to have these and it would form sort super quells the drove you nuts you know Yukon Selebi on the phone trying to untangle the darn thing effective she kind of a nice thing to do because if you're on the phone with the tense conversation or something akin to a task to take your mind off of annoyances that you're dealing with on the phone but anyway so this is called Super coiling and on this is an example of a DNA plasmid which is a sequence of DNA that forms a circle Texas is a nice plasmid DNA and again we really find in this sort of the DNA works completely unwound configuration rather it likes to twist so here's 1 of a little twisted as year's more twisting as even more twist innocent and finally of even the most twisting a case that's really the structures of DNA that we find for short sequences we find it to mount up with itself to form Europeans I showed you that structure 1st for a larger sequences like plasmids we find it's simple quietly and twisting itself up and then for even larger sequences like you care energy arms which I'll show you on the next slide it gets even more twisty than that
peso go on before I get to that here's why it has to get so twisty this is 1 of the benefits of having it coiling up on itself and this is just the DNA in a single equalized self this is a classic picture of that DNA where are you really a tour de force my cross was used to lace the cell and spew out all of its Uday and you could see it's just an enormous amount of DNA for such a small cell and human cells faces similar compaction problem right in the humid you know it would be a what roughly 1 . 7 meters is completely unwound so this property to wonder why on itself to Quayle up with itself is actually a really important 1 and so on
when we look at human DNA and we find that it's compact it up into chromosomes I'm sure hopefully this is not an unfamiliar concept fact actually showed you examples of chromosomes from what I believe is guinea-pigs when we talked about Roman still earlier this this week and in any case so here's some images of human chromosomes over here and on here's how it's impacted us so that the our DNA here's the beginning that we've been looking out over here I admit this is a terrible rendering the user looks like it's a single he likes to note that it's lacking the major Group this is our the blocking the minor groove this is 1 of my pet peeves about artistic depictions of DNA and the slide by at this moment and Enrique so here's the regular B DNA the beating in a scanner wrap around the protein structure called his stone but and this is gonna act like this pools for thread and then his acquire up and those quilts and Aquilla further until eventually you get to something it's nastily compacted into a chromosome I'm not a problem of course and this is the problem with on the phone as well so imagine you're on the phone planners old-fashioned telephone cords which you find that's very hard to untangle the DNA without disconnecting the court making a little break in at a Paris 1 annoying things about supra coiling of old-fashioned telephone cords was similarly with DNA we have is 1 . 7 meter long object and a 20 microns long cell on there has to be a solution to uncoiled the DNA and the solution is to make breaks so short-lived breaks in the DNA using an enzyme called DNA gyrates and on here it's an example of that this is a DH I raised and that a bad debt that's kind of like sisters OK so I notice that it's a dying were the 2 arms down here can open up and I think he just he on to the deal on and then introduce 2 breaks in the DNA allow sooner the superb the DNA to relax stunk oil and then it gets rejoined on this turns out to to be a Achilles heel for bacterial cells for sales in general in other words it's a spot that can be targeted with antibiotics and and we'll be talking about these will be talking quite a bit about the different ways that antibiotics work so antibiotics are pharmaceuticals therapeutics that are given to patients to eliminate bacterial infections or followed actions but in this case and this is a really effective antibiotic that's given quite a bit about this is that the antibiotic Cipro from which reassured many of you in this classroom have taken at 1 point I read somewhere that like 85 per cent of American women come down with the UTI urinary tract infection at some point in the 1st line of of it about accusing insiders often Cipro and other super works by inhibiting the Indian HI arrays of bacteria have here's another 1 as well another inhibitors well OK so
let's talk a little bit more about these bacterial plasmid because I want to transition into a discussion of arms biotechnology and cutting and pasting DNA and large-scale on so oftentimes DNA is transferred amongst organisms using plasmids plasmas are short circular stretches of DNA they need to have all plasmids need to have armed 2 properties must have sequences that occurred to items item number 1 is in origin of replication the origin of replication abbreviated 0 are is the spot that on somehow convinces the the this cell it's taken up the plasmid to start transcribed start on replicating that DNA at a case that origin of replication kicks off replication of the plasmid without that the plasmid would just be there and it wouldn't get copied and would get passed on to the next little guys OK so that's absolutely essential the other essential thing is that the Mannesmann has taken over some advantage on to the new post of pay in other words the bacteria has to take it up and say earlier this is useful otherwise the plasmid will get quickly shunted aside because on cells are under a lot of pressure they have a lot of work to do and they have a limited amount of resources carbon nitrogen oxygen and things like that that are available to you do all of those priorities that they have and that's kind of along with the latest say that this has to confer some of resistance oftentimes to some sort of on about OK so resistance markers are sequences of DNA that encode a protein that confers resistance a case of for for example and you can have any resistance marker that encodes resistant to the antibiotic Texas sight and this gene will work by actively pumping Texas cycling out of the cell OK so when the when the cell takes up this plasmid it's going to synthesize this pot that goes to the surface of the cell and then every time it gets Texas psychologist Continental so furiously and that allows the cell to live so only the cells that have the plasmid will survive an onslaught of the antibiotic Texas cycle in which again is a very common antibiotic that I mentioned a few of you have encountered it's often used for example I believe for acne treatment OK on here some of the classic examples of other antibiotics that are used in my laboratory and other chemical biology laboratories as selection markers for drug resistance and other way this work will cope the sells the bacterial cells on a plate and the plate has an anger which offer you the structure very shortly it's an isolated from seaweed it's basically just a polymer and inside this Aguirre plate will have some concentration of 1 to the about X's and so the only colonies in each 1 the circles as a colony that appears on display are colony hours are bacteria cells that have taken up the plasmid because now those cells are resistant to the antibiotic case so here's another way that this can work so antibiotic that's commonly used as chloramphenicol chloramphenicol inhibits the riders over we talked about the ribosome before the drug resistance gene encodes an enzyme called chloramphenicol as Seidel transferees for cats and this enzyme transfers and Oceano career to a primary hydroxyl of chloramphenicol OK so here's the steel group in a single color they can it's going to get transferred to this primary hydroxyl ions in a reaction that essentially this arms the chloramphenicol preventing it from buying to the ribosome and allowing the cells on this plate of chloramphenicol to live 3rd very common resist marker because section detector cycle and we talked about chloramphenicol 3rd 1 the 3rd 1 are on beta-lactamase antibiotics of this sort notice that this is a beta-lactamase locked him of course is a cyclic ring that has an in and this is beaten because it has 2 carbons alpha beta and so that's the beta lacked that's the origin of the beta-lactam nomenclature which I know we talked about at 51 C hopefully you counted as well in any case on beta-lactam maize is a enzymes encoded by the beta-lactam this gene that that confers the ability to hide July's this on the bond that's part of the beta-lactam rate and on this is a very common gene is found out and environment said you can probably still up you know some dirt over here just outside of Roland Hall and you can readily find this beta-lactamase and so for this reason and medicinal chemists are constantly making new antibiotics that avoid that environmental risk drug-resistant that sort of on the presence a peso for example here are 2 different kinds of being elected beta-lactam based antibiotics and noticed the structural differences this 1 has this Denzel functionality over here this 1 has a carboxyl 8 carboxyl gasses and group instead and so on all those little differences change affect the ability of the drug resistance inside the enzyme conferring drug resistance to to the interbank and idolizes salmon born maybe this carboxyl 8 sticks into the protein and prevents the binding on and that's a useful thing because there were consciously on the hunt for a new antibiotics because they about we have seemed to allow very rapid restore the evolution of drug resistance and so there's a constant need really for our society to develop new classes of antibiotics that are more effective than the previous generation and I am in last 10 years or so there's been a real renaissance of research in this area to develop even more effective antibiotics OK let's get
back to a discussion of DNA structure I showed you structure plasmids here structure of you carry genes are you character you name that's wrapped around nuclear zone the etc. I don't have a much more
say about that let's take a closer look however at the moment the structure of these systems so that his stones are these text Americo teens shown here in yellow and green wearing green fees are positively charged residues of Casillas a arrested is his positive charge to interact with the negatively charged last day as the backbone of the DNA Dodge Charger direction Mason long-range interaction on this wrapping up there so basically hides the DNA and prevents it from being transcribed when it's racked up around his stone it can be read out and that you know how use the transcription and so on basically whether or not the history was wrapping up things as it controls transcription and controls packages so these proteins over here are very tightly regulated as to whether or not they're going to be pointing to the DNA and 1 easy way to do this regulation is to acetylated the lysine side chains of show you that on the next flight OK so 1st of and
this is the structure of lysine lysine has a primary means and here's lysine within a single group but what do you think the charges of something as a primary means at neutral pH which is the pH roughly those of the cell approximately so has has a primary mean functionality a neutral pH What is this charge I'm a very patient died 2 of positive positive period OK good so when so if misused the bearer lysine it will have a positive charge and if it has seal group over here is back to neutral OK so this guy positive charge a group neutral so that controls whether or not that lysine an amino acid of the protein interacts with the DNS it's positive charge it's it's like a homing beacon for DNA right the DNA is negatively charged do these won a stick together if it's acetylated however it's not it's going to be neutral and the 2 are not going to want to interact with each other here's 1 that's even wilder in this case you're taking the primary meaning of lysine side chain and turning it into a secondary or tertiary or even of Wagnerian means and when you do this you're making the lysine side-chain fixed as a positive charge then I should say it's not fixed permanently it used to be thought that it is but now we know that actually this is irreversible modification as is this assimilation OK so this case it's binding to DNA binding to DNA and then when you get methyl groups it's back to the it's still minded to DNA but that it could get acetylated so it's no longer binding to be in case so there's a whole series of different modifications to the surface side chains or the surfaces of the history and all of these modifications have important consequences so for example some of these modifications like these larger ones down here directly his stones into the producer which is basically the garbage disposal for the cell and so those get flushed away and thrown into the trash can and then others like this fast correlation of a hydroxyl functionality found on the surface of the DNA can regulate the structure of the headstone as well and perhaps interfere with this binding to to the negatively charged in OK so all of this stuff is tightly choreographed the enzymes that had each 1 of these of modifications highlighted in blue on the slide and those enzymes are going to control its binding affinity for DNA and in turn control whether or not the DNA is hidden or available for transcription and you can imagine this is very tightly choreographed by the cell if anything gets in there to mess stuff from all kinds of how that can be wrapped right because the cell has to control you know turning on you know specific genes at specific times right you would not want for example you know a muscle cell to suddenly start growing area of neurons or so you know the genes that are required for the neurons grow new right growth or something like that that would be really bad OK so everything is very tightly choreographed and at this level the of the error of her small molecules that inhibit these custodial center lasers I should say of course this is actually a discovery that was made by Jack taught who was a graduate students of when I was registered in the same Lambright was discoveries made when I was registered in the laboratory where I was getting a Ph.D. and short this is what the inner-city lysine surface looks like a bit of the headstone so here is that you have here is an acid heel and then here's Copaxone which looks remarkably like this at lysine this is single life right look very very someone maybe a slightly different number occurrence but it looks very very similar and on so this is going to be 1 possible way to design inhibitors of on enzymes which is to mimic the the substrates effect so this is the starting material for the his stone Diaz said allays the enzyme that shops this is Seidel group and then this compound over here is going to inhibit that the assimilation is more written about this in the book on but in any case these to look very similar and so that the substrate mimicry that that mimicry of the starting material is a very common way to inhibit enzymes it works really well for dynasty that time and again throughout this class OK but I want to change gears now I'm showing you all the cool things that you can build out of DNA I wanna talk to you next about how actually and synthesize the DNA C can build these things OK if you wanna make happy faces or maybe you wanna make the 1st from the faces of DNA that hasn't been done before to my knowledge and you're going to have the know-how to synthesize the DNA said you can make that happen OK so all 1st all talk very briefly about DNA synthesis in allowed but 1st I want to talk to you about DNA synthesis by the enzyme DNA polymerase cases DNA polymerase takes on a a single-stranded piece of DNA called the template and adds a 2nd strand of DNA to that template out all DNA polymerase is found on the planet have a common mechanism in they all require a starting primer strands that gets the
decades it's sort of a running start without this running start the enzyme doesn't know where to begin and this is actually a very useful properties Radio 1 DNA polymerase to come along and start synthesizing random Munir bits and pieces of DNA here and there and it turns out that this is 1 that's been exploited by quite a bit Pinochet some examples of that in a moment OK so this 1 starter is called the primary starting it forms again double-stranded DNA with the targeted 10 plays and Indian a plan raised lengthens this grinding strand in a fight crime to 3 prime direction and other words it grabs onto the street crime and absentee 5 crime etc. a case of this direction here is also common to all forms of DNA polymerase found the planet by Prime to 3 prime disparity materials here .period nucleotide triphosphate and structures so it's a nucleotides that showed you earlier but struck with triphosphate attached to them OK and basically what it's doing is again is taking the greens of Pinehurst strand and extend lengthening it as shown by the scare over here so this is a classic experiment that was done doubt they apply this principle to cracked the genetic code the genetic code is the code by which time sequences D of DNA spell out the amino acids this was in the back in the fifties and early sixties there's this enormous mystery about what the code actually was a case like this you know unsolved major major problem and are Marshall Nuremberg at our locker at In rock or might be run of array of Marshall Nuremberg views this property to crack the genetic code of what he did was he synthesized templates that were long strings of particular of DNA sequences OK so we made a long string of EIS for example and then he looked at me not what was synthesized by by DNA polymerase but downstream was me by ribosomes when you give them a long string of babies and and by doing that he could figure out what the genetic code was OK so at the time again DNA polymerase requires template down to link existing strands and only already plan can start from scratch OK so on a plan racist kind of an exception to this rule DNA polymerase requires a lot of climbing strand reverse transcriptase which takes RNA and so the size of the day we saw that earlier and also requires a primary and dioxin nucleotide triphosphate on REA plan race is kind of a special case by the way any questions yet still freed interrupted the thing comes up because they think it's unclear you want nor information about it don't hesitate to start up a yes this time it to was the primer on that line gets extended but it doesn't get and replicated in the mood that I'm showing you how we talk about PCR will show that it actually can get replicated to help those good question other questions yet 1 yeah education yes but only if OK requested a off some employer like that the names will Williston all and Anthony OK so internees question is how many base pairs of DNA should you have to get the chance uses the primer to get DNA polymerase going OK and on it kind of depends on so you want in a plenary is to pick up a specific gene is a N O a specific sequence of DNA within a complex mixture and so if you want to pick up a specific gene in the human genome you need a primer that a least 18 base pairs in like that it's kind of a magic number of so 18 base-pairs means securing uniquely encoding 1 and only 1 genes in the human genome thanks for asking the question can other here and if you want to do this at you know a lower temperature you can use the Wallace rule and get away with maybe a shorter sequence the OK maybe you don't need such specificity may be a mixed year and year starting our population is less complex had expressed the opinion poll and OK let's move on I'm OK so here is a closer view of what I've been telling you about in hand waiting examples were now zooming down the level of Adams demands that of course is what really thrills me and so here is the the private and DNA polymerase not shown is that of course this primer is forming a base and they are double-stranded DNA To the templated strands and also not shown is that the template strand must have an adenine over here the hybridized to this finding any case the I'm starting material used here is a D Oxy nucleotide triphosphate that it's the Oxy the at the 2 prime hydroxyl and here is the 2 prime are sorry here's the triphosphate functionality the on the demining group in this reaction is going to be died phosphate which is used very commonly in biology is a leading group this is nature's of toss late for me late that you learned about backing 1051 say this thing works really well leaving Europe as well the reasons why we're going to see it quite a bit as a lever grip on the as a DNA polymerase is all of all enzymes that used by bypass as a leading group actually require a dike have confined to this STI phosphate and the requirement is for magnesium in DNA polymerase Caso actually very common problem that I see in my laboratory what when and if he shows up in lab may have trouble the DNA polymerase and 9 times out of 10 it's due to low concentrations of magnesium and a lot of ways to get low-cost stretches magnesium so little tense OK so here's magnesium or what is it doing magnesium is a Lewis acid that's the later To this triphosphate and stabilizing its negative charge doing this makes it a better leading room right this means that if it goes out into the solution a dozen requires a massive rearrangement of water it's already been stabilized its is a lower potential energy that would otherwise be OK so here's the role of DNA polymerase and I'll show you structurally what looks like a moment DNA polymerase brings together the 3 prime hydroxyl of the priming stripped of the primary together at this incoming nucleotide triphosphate and then sets up a nucleophilic attack on the phosphorus of the nucleotide triphosphate No too but there is a 2nd magnesium ions in active sites this 2nd magnesium iron does 2 things number 1 it helps to stabilize the Elcock side formed when this 3 prime hydroxyl was the protonated case and notice that forming an ion-pair relationship with this Alcock number 2 it actually increases the nuclear felicity of the lone pair that's going to do this nucleophilic attack over here OK so what magnesium is doing here it is it's making available better available this moment here for an attack by I'm helping promote the deep-rooted nation of that hydroxide if the hydroxide is the protonated there's more along pair that's available for the tax rate makes sense OK I this forms an intermediate a class of intermediate analogous to what we saw on Tuesday when we looked at the hydrolysis Indian exacts a mechanism that Intermedia is not depicted I'm showing areas over here but again we've looked at that intermediate before so I feel comfortable leaving off of this slide looking into questions about this mechanism that we have a great question and Western neck OK next question is what is amending income from magnesium comes from the food you eat it comes from and you know you added to be tested so typically will add magnesium chloride magnesium sulfate directly to the that the at the door to Tennille tested that we use for these reactions OK but in humans reading in all kinds of food as magnesium and offensive but it's absolutely essential prime state without the magnesium this reaction does not go ahead make sense because I'm showing you want the role it plays OK let's look
structurally ads on what this actually looks like and this is an enzyme that that that again has a number of different author logs for homologs Beazer enzymes that demoralize the same thing reverse transfer prices reverse transcriptase synthesizers DNA from acquiring a template on the enzyme attack is a DNA polymerase that's used quite a bit in research for up to a tool called PCR which all talk about a moment but all of these enzymes have a right-handed structure here's what the structure looks like Casey here's the right here and over here you're my right hand and it's not having on to the unit price of the DNA is red and orange over here and here's the enzyme grabbing onto this unit now what happens is during the synthesis the DNA spreads itself through the clock for my life the and Paul OK and has and then I at a certain that when the cracked nucleotide triphosphate borrowings to the priming strain and the newly synthesized strand of the enzyme can then closed when it closes the palm and the bomb get closer to each other pom-pom fingers OK so close is a little bit like that each time that closes that brings the magnesium is up to the triphosphate setting up formation of the covalent bonds that I showed on the previous slide cases each time the hand closes that's 1 nucleotide that's been out cancel its students rarely have the 1 by 1 cannot stew couple more Bon Bon bond OK now here's the deal this enzyme is really cranking it's actually going to do with thousand up to a thousand of these per 2nd for some reason not cassettes like you to fast food to see really prepared said this enzyme can really turn over very quickly and actually on this is actually something that my lavatories directly observed we actually watched 1 of these enzymes cranking over and we've watched differences as we add different substrates to the enzyme it's really actually fascinating a series of experiments OK so all these enzymes use a common mechanism and again enzyme doesn't close until it gets the crack nucleotide triphosphate that binds to it at that point it closes so in fact actually be rate determining step for this enzyme is actually the arrival of the cracked nucleotide triphosphate based to tease or idea ATP to tease out the CTP to etc OK and I want to tell you a little bit more about why such love with this enzyme this is a dream machine I like fast cars and I like I've this 1 is really amazing said check this out I imagine that a double-stranded DNA was about a meter or so diameter came running along the length of this mystery testing DNA running through the room right and if that
was true idea they :colon race would be about the size of FedEx Oliva truck OK in here including the plan raises there's another replication machinery and I'm leaving offer now that's involved as well but of the roughly the size of that livery truck pulling out right here OK but here's the thing this delivery truck would be racing along at about 375 miles per hour and that's how fast the unit races going in scale to the do you know furthermore it's making about a thousand covalent bonds for 2nd which is insanely insanely fast and In addition haven't talked about this yet but there are other subunits of DNA polymerase that providing an error checking and correction function such that the enzyme is making to scale 1 error every 106 miles or so here which is extraordinary case 375 miles an hour and 1 error every 106 miles this is truly remarkable stuff and you can read more on this reference down here OK now
because this enzyme is so efficient and so superbly specific at getting the right Watson Crick base pairing this is being used as a very commonly in lots lots of laboratories can allows like to about about biochemistry loves forensics laboratories all kinds of labs use DNA polymerase and they often use it to amplify up copies of DNA using a technique called the polymerase chain reaction was invented by Kerry Mullis amongst others the way this works is once you start with some target sequence of DNA shown here in purple again will call the target the template OK so that's the template DNA that you're going to be amplified now there are going to be 3 steps to this PCR reactions in step 1 we heed the DNA of the high-temperature 9 95 degrees and as we've discussed earlier today DNA when it's heated up to high temperature goes from double-stranded to single-stranded it falls apart in stepped to the UN the DAB solution is cool down and that allows the primer as shown here in green and blue to a meal In other words hybridized to be a single-stranded DNA note that these 2 Purple strands don't find each other the concentration of template is very very low effective can get down to just a few copies of DNA so they never find each other like you know lost from each other after the heating step but you have a high concentration of these green and blue primers that can grab on to the correct sequence of DNA that motion targets DNA polymerase drags DNA polymerase to synthesize the specific stretch of DNA and that's done in the 3rd step when of the primers are extended using again DNA polymerase the NTP is magnesium chloride and a temperature of 72 degrees to make this work means a specialty in a plan raised that likes to run at 72 degrees it's a type of Clarice called TAC which is found in hot that's hot springs and it's an organism that's found in these hot springs that has evolved to operate at this temperature and so at 72 degrees the enzyme starts cranking at the lower temperature it's not working at a higher temperature it's not 14 but it's 72 degrees love life again this is Celsius is pretty warm and and it starts emphasizing this black strand of DNA if you do this process a whole bunch of times each cycle you get a doubling of the amount of DNA exceeded this 30 times you get a huge implications for some target template of DNA and target sequence of the unit it makes sense any questions about this I'm hoping I'm not telling you anything you don't already know peace here is now taught at like school level and stuff like that now so OK
summary of right-hand rule we looked at this this is about the magnesium two-plus stabilizing P the nuclear
file and we looked at this already but we move on OK
so DNA polymerase is also a terrific target for inhibitors and reverse transcriptase inhibitors have been very very important compounds for for stopping synthesis of DNA this many reasons why it wanna stop the synthesis of DNA and to treat for example cancer where cells are dividing uncontrollably if you can shut down the replication of DNA you have an effective way of stopping cancer and effect actually childhood leukemias were stopped in their tracks back in the late 70's and through the wonderful research of 1 of my scientific heroes the great Gertrude shown here Gertrude and was about born in 1918 her parents wanted her to become a nurse case of a senator college they said they will become a nurse she actually wanted to become a chemist and and sigh when World War Two broke out she was given the opportunity OK so during World War Two men were sent to the front to fight and there are a lot of opportunities that were available to women that weren't available before that and she's 1 of those people who took that opportunity she joined up Burroughs Wellcome where she worked with George Hitchings for her entire career she's 1 of these people spent her entire career at a single company and on together at George Hitchings she discovered this class of compounds that inhibit DNA polymerase and ended up actually having a major impact on childhood leukemia and she's here she's a true superstars sites 1 last thought that nurtured Ellie and never received her Ph.D. she went on to receive a Nobel Prize in the eighties for this work and she did it through sheer force of will through her determination to contribute something and I highly highly recommend an interview of her and all even put it up on the board over here there's if you want to learn more about her
this is terrific interview of her that's been on the
documentary that I recommend case of a documentary is called up and Isaac can you see this OK Natalie OK Isaac the Newton and need for and the director is 1 of the great Michael Apted I could actually have a whole class just on Michael Apted's documentaries but in any case on she's interviewed in the documentary and she talks about there are incredible pride and just joy that she felt which you'd visit children's hospitals and she would see kids being treated for the 1st time with her compounds and on how transformative that was in the life of these kids these are kids units slated to die at that very young ages and ages of 10 and 12 and there were some getting cured by these contents they let's take a closer look and understand how these compounds work OK so
she invented a series of bomb inhibitors of and DNA polymerase that look like that's OK so this is 1 called Easy tea it's also used varied as they are In tight HIV compound because it inhibits reverse transcriptase it looks kind of like a DNA-based it has a dioxin at the 2 prime hydroxyl but in place of the 3 prime hydroxyl there's an easy ride case so what happens is this gets taken out and fast correlated to give a triphosphate and then DNA polymerase attempts to use it as a substrate for what ends up happening is instead of a three-time hydroxyl there's an easy here and the invited caps the synthesis the under the new strand of DNA preventing it from being one-third of similarly this is DTC another compound that's used in the treatment of HIV and also leukemia and instead of a prime hydroxyl it has a hydrogen there and so again it gets used by DNA polymerase and the point raised no longer the nieces strand of DNA case of both the shutdown DNA synthesis on simplifying things a little bit there's another class of compounds known nucleoside analogs is nucleoside analogs that are also used against HIV and but a decade gives you have a taste for look at your daily that she had a major major impact in the fight against viruses and in the fight against leukemia and that I actually had the pleasure of meeting her once in my life on the day I was getting my PhD at my graduation she was receiving an honorary PhD from Harvard and I just shook her head and that's about it and because I didn't have any profound conversation with the richest my regret but I truly remarkable woman true superstar science but I can't say enough about have lecture about her and when we move on OK so
that's a synthesis in cells 0 question over here the of this is such a good question and so what U.S. what on problems OK so bottoms question is how do you get the compound to the cancer cells what we're going to see time and again is that cancer cells are actively eating up everything every little bit of a nutrient that they could fight they will just be devouring stuff that surround them and so for this reason they and other dividing cells will more preferable preferentially take up drugs like these that are fed to the patient the injected into the patient OK so the problem of course is that the other cells in the body that are also dividing and Noel unfortunately take up these chemotherapeutic and also end up dying because their DNA synthesis will be impaired OK request and thanks resting on a case said I wanted I don't have religious about chemical DNA synthesis I think it's absolutely amazing topic and this is 1 of those areas of organic chemistry that is a true triumph of peso and we're going from strength to strength today in DNA synthesis in the laboratory has been so optimized and there were at the point where we get 99 . 9 per cent yields for reactions and this is really the ultimate goal in the quest to do organic synthesis and it was set in line by the great building Cronin who won a Nobel prize but also Bob Wessinger and money for others these guys deserve a Nobel Prize because this really dig kick off of a revolution by technology that was made possible by the synthesis of DNA to make primers which in turn allows PCR which in turn allows smiley faces out of DNA and all those other great discoveries that we've been talking about can I just want you assume this topic in the don't get
too worked up about it in the end we have these machines that look like this where you have a bunch of bottles down here that injects that you can use it to program a computer up here do it open to inject on regions directly into flow cells that have the DNA said that a sequence that you're trying to synthesize so you do this using solid support base emphasis on where did the Wiese strand of DNA is the is attached to some sort of and you basically flow in the regions 1 after another and couple the correct nucleotide directly onto the DNA sequence it's a little more complicated than that but here's what you need to know if you want a synthesized any sequence of DNA that 150 base pairs a 150 basis or less you get on the Web and you call what someone I'm probably Texas or somewhere like that and there will be a whole warehouse of machines like this and you enter into some form on the way on this on their website exactly the sequence of DNA that you want and that sequence will get get ported to a machine that looks like that's warehouse just filled with these machines and there's a bunch of arms technicians on roller blades that are going to be running around in keeping the machines that with 3 agents you won't see any of this because at the end you get that package with your DNA sequence probably in a couple of days some of these I think even overnight raids on so overnight you're going to get your sequence of DNA perfectly cleaned up purified delivered to you of 19 5 per cent a 99 per cent purity depending on how much you decide to pay for it and and I you know it'll be perfect every time you won't even have to think about the chemistry I will that's the goal of organic synthesis the goal of organic synthesis to make this totally turnkey so that we can then use this data to answer biological problems which is what I want talked about next OK here is 1 example of using this amazing ability to do DNA synthesis to address biological problems you can print sequences of DNA on microscope slides OK so here's a machine that's nothing more fancy there I I think in this case is it inkjet printing device again it's going to be printing out Little Leaguer nucleotides 1 after another on these identical microscope slides so each square down here is a microscope slide of across the surface of microscope slides were going to have a bunch of different sequences of DNA that had been printed down onto specific spots so what this is going to do is this is going to give us an array of different sequences of DNA each 1 capable of hybridizing forming Watson Crick base period to a different other sequence of DNA or RNA OK this is good technique called the DNA microarray sir
here's the way this works for me to show
you what it looks like OK so now I'm zooming in on the microscope slide OK so here's what it looks like each spot over here is of a different sequence of DNA and he set up the hybridization such that and you end up with the other 4 were simply labeled sequence OK so each again each spot and has a different sequence of DNA if the if there is a complementary sequence in your sample then you will see fluorescence so ingredient that tells you that your sample has this particular sequence OK and it's known exactly what the sequences in green that's down there can be achieved USA besides the complimented put down right there a case of I don't know what to say this is the gene that confers resistance to brittle beta-lactam antibiotics yes you have that gene because you're seeing green spots right here in practice this can be made a lot more complicated OK let's imagine now that we have 2 samples 1 that we label with red sequences and 1 that we label with green sequences In this allows us to compare all of the different DNA sequences present for RNA sequences present and comparing them against Red vs green OK and let's take a closer look OK
so each Lula Kotite hybridize is to a different MRA transcripts were aware of the 1 sample is labeled ingredients and the 2nd samples labeled and red OK
here's the latest works on sample wines use reverse transcriptase all these on day into Indiana and you add the green floor for in sample to use reverse transcriptase and label all those with a red floor for OK so sample 1 is green simple too is red and then you add both of those to the DNA microarray the ones that are agreeing are telling you know that she is up-regulated those types cells the ones that Arad is telling you it's up regulated the other types of cells and were ready green overlap you see yellow OK so this allows you to compare experiment versus control and the possibilities are endless for example you can look at cancer versus non-cancerous cells virus-infected versus normal G 1 phase of the cell cycle bosses estate's all explain these phases in moment young cells forces of old Dr untreated versus Notre etc. and so in it and you get these massive erased where you get a huge amount of data each 1 of these red spots that could tell you that so that a gene that up-regulated in say cancer cells a virus-infected cells and then the yellow ones those the ones you don't have to worry about the green ones that right because that's the same in both types of cells the green ones however are ones that are up regulated in and b let's say the non virus cells a came down regulated in the case of the cancer cells to you can see in 1 very simple experiment relatively simple little complicated you get out the enormous amount of information about the transcription activity of an entire self again this is also replaced by DNA synthesis of chemical DNA synthesis OK so here's an example of this is specific example of example I'm going to use it is on the small-molecule FK 506 and also called took all of us this is a immunosuppressant that was discovered by the Chemical Co the pharmaceutical company Fujisawa hence the name FK but as new suppressants is given to patients who I had an organ transplant case so this was a stick toss a revolution in the area of liver transplantation for example when it 1st became available in the 80's Ladies and early nineties OK so if you give the structure patients who have just had a liver transplant they will not reject the new transplanted liver came because the immune system suppressed you can see the problem with that approach the item in the new system is suppressed that means that if you get a cold in big trouble setting out a side Canada's other things that you can let's try to look at what pathways a change when we feed themselves this compound in doing so we can start to identify the pathways associated with the immune response and
said and here's a classic experiment done by the company called Rosetta infamous for for Maddux now Merck Pharmaceuticals and In this experiment they have 2 kinds of cells 1 kind of cells are green but they were treated with the green of flora force the transcripts attribute the green floor 4 and in the red cities have no drug added to them and red those transcripts were treated with the compound that should on the previous cycle 55 6 In yellow there's a whole bunch of different compounds check this out in green over here this is a can't-miss as they approach This is a gene that encodes a protein that must be turned off when the compound is added a paid notice that informs the bright green spots over here here's 1 that gets turned on it forms a red spots the yellow and orange ones would not worry about those so much apparatchik imagine doing all kinds of analysis of the stuff and the bioinformatics either these things the computation the allies of stuff fascinating affair really exciting area of outcome computer science research OK does this make sense 1 experiment you get the whole pathway that's being targeted by this drug the I'm not tell you very much about the pathway now need will talk about it later yeah question about but the OK yes so In practice you have a laser that scans across the surface of this my parade and then you have a CCD camera that captures the intensity of each spot the attack I'm hoping this is blow your mind because certainly blows my mind OK so now I want to change gears a little bit and talking about how to analyze the DNA sequences of DNA and before I deal with the at the time here on a run all the way up to the 10 before the hour but I just want to tell you I have some good news for you a little we can treat thanks for bearing with me of the quiz the midterm will only cover through Chapter 3 will be no chapter for the mid-term come comeback on Tuesday will be talking more about jumped out there ready to go or not gonna stop now they just won't let know that I will be talking more about DNA and then I will go into or enable the mid-term and next Thursday a week from today will only cover through Chapter 3 of the current with the batter discussion I want to switch gears very slightly earlier alluded to Padaro switches used in those dish plates so showed you earlier on agarose has the structure down here it's isolated from seaweed and it for it is a polymer that is tightly Crosslake deformed enact case could actually form little bricks of formed the stuff and then you can apply the DNA 2 1 end of the agarose gel so here's your little brat call that agarose gel and you attacked you your DNA to 1 end of this gel and then use electrophoresis to punish the DNA through the polymer they recall that DNA as negatively charged so it will be attracted to the positively charged terminal will to possibly charge electrode that cathode at 1 end of the year electrophoresis apparatus appears again you have these wires coming out they're going back to some other source of electricity some power supply and that's pushing the DNA through the gel the DNA will get separated out there and on the basis of its size OK so big a DNA is going to get cannot caught up in this complicated network over here wears a little pieces of the day we are going to find it easier to flow through these little Porter's so practice what this looks like is this I think British only 1 of these Iris jails this is the top of the jail where the big pieces are here's the bottom of the gel with a little pieces are again the big pieces have not migrated as far because they got stuck in the interest issues of the DNA frequently we add what's called a DNA letter to 1 lane of the gel and that provides basically a ruler tells us what sizes of DNA were look How was visualized where these DNA pieces in this color over here up where we had to the jail yes in Bromide Takeo yet we've added diet .period bromide that concentrates the in the DNA and as fluorescence his works really well this tells us a lot about the late late and a little bit about its structure you can look at for example super coiling using this technique OK I guess here is that the bromide here is is interpolated into DNA and this practices what it really looks like it has a bright purple color In addition to
separating on jails we very commonly separated out DNA using capillary electrophoresis and this is a much more effective way of separating out DNA that differs by a single base inside take a large number sequence the DNA and then seperate them out loud said such a view have you know 100 birds 99 murders 1997 rose 96 etc. all neatly separated out using this technique called capillary electrophoresis it's the same electrophoresis technique but with a different kind of mobility layer of different mobility phase but it's basically using electrophoresis again but in a capillary OK here's why this is important and you can use this Gelb separation as a way of sequencing DNA if you had some way of breaking up into little pieces were each piece differs by 1 base pair In practice what we do it is on and I will only tell you about this 1 over here OK this is the old-fashioned way to do this and in using these actually use acrylamide gels is back when I suppose stock but will no longer be this no 1 my life this way we all do it this way here's the latest works and what we do is we had a very low concentrations of died the Oxy terminated so this is exactly what was invented by Gertrude Elliott and the Gertrude Ellie inside that I showed you earlier on this is missing the 3 prime hydroxyl and has a hydrogen and place over here and on the street crime was Terminator is missing this hydroxyl shut down DNA polymerase synthesis when they do they also carry along a Florida for that has a specific wavelength associated with the case you add a 1 percent concentration of the odd died at sea see inhibitor that has a green floor for you at 1 per cent but that has G and has a red floor for you at 1 per cent that that has she ever has a deep purple for and you see where this is going to have a word idea Terminator is each 1 with its own floor for use at these 4 floorboards up so there's no overlap of the wave likes a little overlap as you can get in there and you separated out the sequences using capillary electrophoresis and what you can do is actually read out based upon the red green or blue status of each of those guys that you have a sequence that says she HEC TT GTG etc. and a practice we have to be unit simply take the data feed into a program and the computer gives us a sequence that has stuff is masterly automated fact we're at the point where allowed simply sends out the sequences and there's another lab accused me campus but now it's actually worth it's here in San Diego OK assets in Sandiego that does all the sequencing for us in a costly few dollars per sequence and that's that prices dropped enormously OK any questions about anything we see today a gap in the law I don't think it's going to happen Oh yeah yeah uh was put that off Abkhazia fluorescence technology which there at the
last thing you can use DNA technology for all kinds of things that used very extensively you can even use it onto program changes in organisms like dress-up a lot and you can use this to test hypotheses a little hard to tell the you see the extra eyeballs they're growing out of this organism Bible at the start of all involving the 3 had actually program you can insert sequences of D-Day into these organisms and coax them to get turned on at specific times and use this test and whether or not we understand what specific genes do and this is actually a fascinating story that actually deals with the heat shock protein OK
and there's a couple of ways of modifying the DNA in organisms 1 way is to do it randomly using compounds like this 1 this mechanism will talk more about on Tuesday so let's stop here when we come back next time will be talking more about this


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