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© Department of Chemistry, The University of Western Ontario
Chem 2223b Intersession 2008: Nucleic Acids
• This chapter focuses on some of the biological and chemical aspects of nucleic acids, specifically DNA. We will examine its properties, sequencing, and laboratory synthesis.
• Background material, from Chem 2213a or otherwise, that is important includes:
o Alcohols
o Amines
o Strengths of acids
o Substitution and elimination reactions
• After studying this section, attempt all practice problems on nucleic acids.
Nucleic Acids 2
A. Structure and Composition of Nucleic Acids
• Nucleic acids, which include DNA and RNA, are a class of biopolymers that carry genetic information. They can be sequentially hydrolyzed by specific enzymes or by chemical means (acid).
Polymeric nucleic acid
Mixture of nucleotides
Mixture of nucleosides + phosphate
Mixture of nitrogenous bases + phosphate + sugar(these are the products of total hydrolysis)
RNase or DNase
various phosphatases
various nucleosidases
Enzyme hydrolysis is very selective.
Acid hydrolysis is much more difficultto control and can often result in the totalhydrolysis of the nucleic acid.
Nucleic Acids 3
1. Nucleobases (or simply “the bases”)
• Nitrogen atoms 1 and 9, in pyrimidines and purines respectively, are used to form N-glycosidic linkages with sugars (ribose or deoxyribose).
Nucleic Acids 4
• With all of the five, tautomerism is possible, and the structures shown are the predominant forms. Interestingly, when Watson and Crick worked on the structure of DNA, they used the structures found in old textbooks, which showed the enol forms.
• Tautomerism involves only the rearrangement of H and the bonds. Some examples:
N
NH
NH2
O
N
N
NH2
OH
NH
NNH
N
O
NH2
N
NNH
N
OH
NH2
C (keto form) C (enol form)
G (keto form) G (enol form)
Nucleic Acids 5
2. Nucleosides
• Nucleosides are the bases attached to sugars (ribose in RNA, 2-deoxyribose in DNA) as β-N-glycosides (N1 pyrimidines, N9 purines).
CHOOHHOHHOHH
CH2OH
OHHOH2C
OH OH
O
CHOHHOHHOHH
CH2OH
OHHOH2C
OH
O
D-Ribose 2-Deoxy-D-ribose
NHOH2C
OH OH
O
N
NH2
O NHOH2C
OH
O
a ribonucleoside a deoxyribonucleoside
N
N
NH
O
NH2
Nucleic Acids 6
3. Nucleotides
• Nucleotides are composed of nucleosides and one or more phosphates.
• Phosphates can be attached to one or more of the OH groups (2’, 3’, 5’).
• The “prime” refers to the numbering of the sugar.
NOH2C
OH
O
N
NH2
O
deoxycytidine-5'-monophosphate
PHO
O
O
NOH2C
OH
O
guanosine triphosphate (GTP)
N
N
NH
O
NH2
OH
P
O
O
O
POP
O
O
HO
O
O
Nucleic Acids 7
4. Nucleic Acids
• Nucleic acids are phosphate diesters of nucleotides. The structure has two anti-parallel strands in a right-handed helix. A-T and G-C base pairing by H-bonds.
5’-endN
N N
NN
OH
N N
O
N
H
H
NH
H
N
NN
NH
N NH
O
CH3CH3O
H
N
NN
NOH
N
NO
N
H
H
NHH
N
NN
NNH
N
N
H
O
CH3CH3O
H
O
OP
HO
O
O-O-
O
OP
O
O-O-
O
O
OP
O
O-O-
O
O
HO
O
OHO
PO-O-
O
O
OO P
O-O-
O
O
O OP
O-O-
O
A
OT
O
G
OH
C
O
3’-end
Nucleic Acids 8
• The base pairs, AT and GC, occupy the same amount of space, so the right-handed helix is usually smooth and uniform. This arrangement also produces two grooves that are different in size, termed the major groove and the minor groove.
• Proteins, drugs, and other molecules can take advantage of the functional groups present in these grooves and selectively bind to them.
• Other binding methods include:
o Non-selective interactions with the negatively charged phosphate ester backbone
o Intercalation, where a molecule intercalates (inserts itself) between base pairs. This is seen with some drugs or carcinogens, where they bind by hydrophobic and dipole-dipole interactions.
N
NN
N
NHH
H
CH3O
O
N N
NN
N
O
N
NH
H
H
H
HN
O
NN
A
T
C
G
minor
minor
major
major
Nucleic Acids 9
A protein bound to the major groove
Nogalamycin intercalated (one strand shown for clarity in the right diagram)
Netropsin bound to minor groove
Nucleic Acids 10
B. DNA Sequencing
• Molecular biology and genomics, two fields that involve the study of genes and genomes, underwent explosive growth in the last 10-20 years. This growth would not have been possible without a method to sequence DNA rapidly and accurately.
• Sequencing methods were developed in the 1970’s by Sanger and Gilbert, but automated instruments did not appear until the late 1980’s. This instrumentation, invented by analytical chemists, made the Human Genome Project possible.
o Sanger won two Nobel Prizes in chemistry for his work on two biological problems: peptide (insulin) sequencing in 1958, DNA sequencing in 1980.
• One important property of DNA is fundamental to sequencing. At high temperature, DNA melts/denatures into single strands that anneal upon cooling.
5'‐GCTATCGGATCG‐3'3'‐CGATAGCCTAGC‐5'
Double-stranded DNA(H-bonded base pairs)
5'‐GCTATCGGATCG‐3'
3'‐CGATAGCCTAGC‐5'+
Single-stranded DNA
heat (melt)
cool (anneal)
• This behaviour allows DNA to be replicated in the lab…
Nucleic Acids 11
1. Replication by PCR
• In 1983, Kary Mullis, a chemist working for a biotech firm in California, decided to tour the Pacific Coast Highway on his motorcycle. It was during that trip that he conceived an idea for the rapid amplification of DNA. (Nobel Prize in Chem, 1993)
• Known as the polymerase chain reaction (PCR), billions of copies of DNA can be made using the enzyme DNA polymerase. The principles of PCR are also used in DNA sequencing, so it is crucial to understand the fundamentals of PCR first.
• DNA polymerase uses a strand of DNA as a template and synthesizes the complementary strand using the four 2’-deoxyribonucleotide triphosphates (dNTPs = dATP, dTTP, dCTP, and dGTP).
o The direction of synthesis by the enzyme is 5’ 3’ and is performed by linking the 5’ end of a dNTP to the existing 3’-OH end of the growing chain.
baseOH2C
OH
OPO
OO
POPO
OHO
O
O
GrowingChain-OH3'5'
OH2C
OH
OPO
OO
GrowingChain base5'
new 3'OPOP
O
OHO
O
O
Nucleic Acids 12
• Since DNA polymerase requires an existing 3’-OH group, a small fragment known as a primer is required. After annealing, elongation can occur
o Note: there are TWO templates, so two primers (forward + reverse) are used.
o Biologically, the primers are synthesized by the enzyme primase. In the lab, we can simply include some carefully designed primers in the reaction mixture.
3'‐CGATAGCCTAGC‐5'
5'‐GCT‐3'
melt
3'‐AGC‐5'
5'‐GCTATCGGATCG‐3'
dNTPsDNApol
elongate
5'‐GCTATCGGATCG‐3'3'‐CGATAGCCTAGC‐5'
5'‐GCTATCGGATCG‐3'3'‐CGATAGCCTAGC‐5' 3'‐CGATAGCCTAGC‐5'
5'‐GCT‐3'
5'‐GCTATCGGATCG‐3'3'‐AGC‐5'
5'‐GCTATCGGATCG‐3'3'‐CGATAGCCTAGC‐5'
annealprimers
Nucleic Acids 13
• The cyclical nature is well-suited to automation and is the basis of PCR. To replicate DNA, the PCR procedure requires these components:
o DNA polymerase isolated from Thermophilus aquaticus (Taq)… why Taq?
o DNA to be replicated, both forward and reverse primers, dNTPs, buffer
o Temperature-cycling equipment (thermocycler or multiple water baths)
• One cycle consists of three steps, e.g.:
1. 94 °C for 30 sec (melting)
2. 54 °C for 30 sec (annealing)
3. 72 °C for 1.5 min (elongation)
• After 35 cycles, there are 235 copies (35 billion).
Nucleic Acids 14
2. Sequencing by Chain Termination
• The chain-terminator strategy is a popular method for DNA sequencing and also uses DNA polymerase. Some major differences from PCR:
o Only the forward primer is required. Because the DNA strands are complementary, it is sufficient to sequence only one strand.
o All four dNTPs are present, but the mixture is also supplemented with a small amount of 2’,3’-dideoxyribonucleotide triphosphate (ddNTP)
• Since ddNTPs do not have a 3’-OH group, they act as chain terminators. If a ddNTP is incorporated into the growing chain, elongation stops.
baseOH2C OPO
OO
POPO
OHO
O
O
GrowingChain-OH3'5'
OH2C OPO
OO
GrowingChain base5'
No 3'-OH, cannotadd next nucleotide
• The reaction mixture contains both dNTPs and ddNTPs, so the termination point is essentially random and results in product DNA chains of assorted lengths.
Nucleic Acids 15
a. Chain-termination method 1: radioactive ddNTPs
• This method requires four separate reactions, one for each base (A, C, G, T). All dideoxy compounds are 32P-labelled on the P attached to the 5’ oxygen, and that phosphorous is the one incorporated into the chain.
baseOH2C OPO
OO
POPO
OHO
O
O
GrowingChain-OH3'5'
OH2C OPO
OO
GrowingChain base5'
No 3'-OH, cannotadd next nucleotide
* *
• Each reaction contains the DNA to be sequenced, forward primer, DNA polymerase, all four dNTPs, plus a small amount of one of ddATP, ddCTP, ddGTP, or ddTTP.
o A reaction: all four dNTPs + some ddATP
o C reaction: all four dNTPs + some ddCTP
o G reaction: all four dNTPs + some ddGTP
o T reaction: all four dNTPs + some ddTTP
Nucleic Acids 16
• Therefore, in the “A” reaction, termination is only possible after an A and occurs only if ddATP is by chance incorporated, resulting in a radioactive chain. In the example below, three new chains are formed, two of which are radioactive.
3'‐CGATAGCCTAGC‐5'dNTPs
5'‐GCT‐3'
ddATP*
Template 5'‐GCTA*‐3'5'‐GCTATCGGA*‐3'5'‐GCTATCGGATCG‐3'
• The same occurs for the “C” reaction. Note that the first C present in the products is never labelled, because it was added into the mixture as a primer.
3'‐CGATAGCCTAGC‐5'dNTPs
5'‐GCT‐3'
ddCTP*
Template 5'‐GCTATC*‐3'5'‐GCTATCGGATC*‐3'5'‐GCTATCGGATCG‐3'
Nucleic Acids 17
• For the “G” reaction, three radiolabelled chains are formed.
3'‐CGATAGCCTAGC‐5'dNTPs
5'‐GCT‐3'
ddGTP*
Template 5'‐GCTATCG*‐3'5'‐GCTATCGG*‐3'
5'‐GCTATCGGATCG‐3'5'‐GCTATCGGATCG*‐3'
• Finally, the T reaction…
3'‐CGATAGCCTAGC‐5'dNTPs
5'‐GCT‐3'
ddTTP*
Template 5'‐GCTAT*‐3'5'‐GCTATCGGAT*‐3'5'‐GCTATCGGATCG‐3'
• Every radioactive chain is of a different length, allowing separation by size using gel electrophoresis. Photographic film is then placed over the gel, and the decay of 32P exposes the film, forming a band that corresponds to the location of the chain.
Nucleic Acids 18
A reaction5'‐GCTA*‐3'5'‐GCTATCGGA*‐3'
C reaction5'‐GCTATC*‐3'5'‐GCTATCGGATC*‐3'
G reaction5'‐GCTATCG*‐3'5'‐GCTATCGG*‐3'5'‐GCTATCGGATCG*‐3'
T reaction5'‐GCTAT*‐3'5'‐GCTATCGGAT*‐3'
A C G T
wells
• Since the primer is known, the sequence can be deduced from the bands. This also allows the determination of the original 3’ 5’ strand.
• Major disadvantage: four reactions are required.
Nucleic Acids 19
b. Chain-termination method 2: fluorescent ddNTPs (dye-terminator method)
• ddNTPs are tagged with four fluorescent dyes, one for each ddNTP. Each dye type, and hence ddNTP, has a different wavelength (colour) of maximum fluorescence.
• For the four dyes shown here, the wavelengths of maximum fluorescence are: ddATP = 560 nm, ddCTP = 595 nm, ddGTP = 535 nm, and ddTTP = 620 nm
Nucleic Acids 20
• It is therefore possible to distinguish the chain terminators by measuring the fluorescence wavelength, so only one reaction, not four, is required. The reaction mixture contains all four dNTPs and all four dye-linked ddNTPs.
3'‐CGATAGCCTAGC‐5'
dNTPs5'‐GCT‐3'
ddNdyeTPs
Template
5'‐GCTAdye‐3'
5'‐GCTATCGGATCG‐3'
5'‐GCTATdye‐3'5'‐GCTATCdye‐3'5'‐GCTATCGdye‐3'5'‐GCTATCGGdye‐3'5'‐GCTATCGGAdye‐3'5'‐GCTATCGGATdye‐3'5'‐GCTATCGGATCdye‐3'5'‐GCTATCGGATCGdye‐3'
λmax emission
535 nm595620560535535595620560
Adye = 560Cdye = 595Gdye = 535Tdye = 620
Sequence:
Nucleic Acids 21
• Moreover, the separation can be automated by capillary gel electrophoresis. Instead of loading the sample onto a slab of gel, it is loaded into a gel-filled, glass capillary.
• Loading is done by dipping the capillary in reaction mixture for a few seconds.
• Dye-labelled chains travel along and are detected by fluorescence at the other end of the capillary.
reactionmixture bufferbuffer
laserexcitation
fluorescencedetector
sample gel-filled
capillary
Nucleic Acids 22
• The shortest chain arrives at the detector first, and its wavelength of fluorescence is then correlated with the identity of the dideoxy base. Recall that each dye-labelled base has a different emission wavelength.
• A plot of fluorescence as a function of time affords the following:
time
fluor
esce
nce
inte
nsity
Adye = 560Cdye = 595Gdye = 535Tdye = 620
560620 595 535
535560
620595 535
• By examining the order of the appearance of fluorescence at the four wavelengths, the sequence can be deduced: 5’-(known primer)-ATCGGATCG-3’.
• Remember, the sequence of the original template is the complement of the above.
Nucleic Acids 23
3. Pyrosequencing
• Pyrosequencing, also referred to as sequencing by synthesis, is a newer technique that does not rely on chain termination. No dideoxy compounds are used.
• Instead, pyrosequencing relies on the detection of the pyrophosphate that is released during the DNA polymerase reaction. Using a cocktail of reagents and enzymes, the production of pyrophosphate is detected as light emission.
baseOH2C
OH
OPO
OO
POPO
OHO
O
O
GrowingChain-OH3'5'
OH2C
OH
OPO
OO
GrowingChain base5'
OPOPO
OHO
O
O
detected as light
dNTP
• The concept of pyrosequencing is straightforward: if the wrong dNTP is added to the mixture, no pyrophosphate is formed, and thus, no light. If the correct dNTP is added to the mixture, pyrophosphate is made, and light is emitted.
Nucleic Acids 24
• The sequencing cocktail contains one dNTP, adenosine 5’-phosphosulfate (APS), luciferin, and the enzymes luciferase, ATP-sulfurylase, and apyrase. This carefully designed four-enzyme system works in a cascading system.
• The conversion of pyrophosphate into light involves two enzymatic reactions. In the first reaction, ATP-sulfurylase uses APS and pyrophosphate to make ATP.
Ade
OH
O
OH
P
O
O
O
SO
O
O
OPOPO
OHO
O
O
O
Ade
OH
O
OH
P
O
O
O
POP
O
O
HO
O
O
O
APS
ATP
OSO
O
O
Nucleic Acids 25
• In the second reaction, luciferase uses ATP, luciferin, and atmospheric O2 to generate oxylucerin in its excited state, which releases light upon relaxation... a mechanistically very complex reaction and an example of chemiluminescence.
Nucleic Acids 26
• Thus, if the dNTP is added to the growing chain, light is produced, and the light can be recorded and quantified by the instrument. If no light is released, then the dNTP in the mixture must not correspond to that of the correct sequence.
• To allow this method to work in a cycle, apyrase destroys leftover dNTP.
• The next dNTP is then added, and the reaction monitored for light release.
Nucleic Acids 27
• The amount of light released by the luciferase reaction is directly proportional to the amount of ATP made, which is in turn proportional to the amount of pyrophosphate and hence, the amount of dNTP added to the growing chain.
• A pyrogram is a plot of light emission as a function of the nucleotide added. The relative peak areas are proportional to the amount of dNTP added to the chain.
Nucleic Acids 28
C. Chemical Synthesis of Nucleic Acids
• Like the modern method of peptide synthesis, DNA synthesis is also performed using a solid-phase approach. The 5’-OH and 3’-OH ends need protecting groups.
• We also need a way to condense the phosphoric acid and the alcohol to make a phosphoester. (This is not a carboxylic acid ester, so DCC does not work well).
BaseO
O
PO
O
HO
O
BaseO
OH
HO
BaseO
O
PO
HO
OBase
O
OH
O
condense
5'
3'prot
ectin
g gr
oups
requ
ired
Nucleic Acids 29
1. 5’-OH Protecting Group
• The 5’-OH group is protected as a dimethoxytrityl (DMT) ether. Such a protecting group can be easily removed SN1 using trichloroacetic acid (TCA).
OR
OMe
MeO
OMe
MeO
R = 5' end of nucleotide
HHOR
• Some thoughts…
o Why are we using dimethyoxytrityl, not a trimethoxy or a non-methoxy?
o Does the byproduct formed from deprotection absorb visible light?
Nucleic Acids 30
2. 3’-OH Protecting Group
• The 3’-OH group of the first unit is linked to a polymer with a COOH group using DCC, forming an ester. Recall how the amino acid COOH end was linked as an ester in peptide synthesis, where it served as a means of attaching the compound to the insoluble polymer.
• The ester is not destroyed by the TCA used to remove the DMT protecting group.
• It is also necessary to protect the functional groups present in the purine and pyrimidine bases, but we will not examine the details.
• Once the first unit is attached to the polymer, its DMT group can be removed using TCA, and the second monomer can be added in a coupling reaction.
BaseO
O
DMT-O
O
BaseO
O
HO
O
Nucleic Acids 31
3. Coupling Reaction
• Although our final product needs to have a phosphodiester bond, the phosphodiesters are not made directly. Instead, we use a phosphoramidite (PIII oxidation state) and then oxidize it to a phosphate (PV state).
• This is simply an acid-catalyzed substitution, where the amine is replaced by an alcohol (which happens to be the 5’-OH of the attached monomer).
BaseO
O
P
DMT-O
ON
Acid
R-OH
BaseO
O
P
DMT-O
ORO
N,N-diisopropyl-β-cyanoethyl-phosphoramidite
CN CN
• The acid used must be chosen very carefully: it must not remove the DMT group
Nucleic Acids 32
• In this instance, the acid used is tetrazole. At a first glance, it appears to be a base, but because there are many electron-withdrawing, sp2-hybridized nitrogens present, the compound has a relatively low pKa (~5) and can act as a weak acid.
NH
N
NH
NN
NH
NNN
NH
pKa 17.5 14.5 9.5 4.9
tetrazole
• After the coupling reaction, the phosphite needs to be oxidized to PV.
BaseO
O
P
DMT-O
ORO
CN
BaseO
O
P
DMT-O
ORO
CN
I2 / H2O
O
• What is the purpose of the cyanoethyl? If it were not there, the phosphate O− could potentially act as a nucleophile. It is not removed until the desired length is reached.
Nucleic Acids 33
BaseO
O
HO
O
BaseO
OP
DMT-O
ONCN
+
NNN
NH
add
BaseO
O
O
O
BaseO
OP
DMT-O
OCN
1. Coupling
2. Iodine
BaseO
O
O
O
BaseO
OP
DMT-O
OCN
OBaseO
O
O
O
BaseO
OP
HO
OCN
O
Repeat cycle
OR
Cleave fromresin andremove thecyanoethyl togive product
3. TCA
Nucleic Acids 34
• The cleavage of the product from the insoluble polymer and the removal of the cyanoethyl groups are both done together using ammonia in water (NH4OH).
BaseO
O
O
O
BaseO
OP
HO
OCN
OBase
O
OH
O
BaseO
OP
HO
OO
O
NH2
NH4OH
• The cyanoethyl group is lost in a β-elimination reaction (E2).
• Why not simply use a strong acid to hydrolyze the ester? In peptide synthesis, we used HBr in acetic acid. Why is HBr unsuitable here?
Nucleic Acids 35
D. Unintentional Damage to DNA
• DNA can be damaged by UV light, carcinogens, and other environmental factors. In all cases, DNA damage can lead to cancer, mutations, and cell death.
1. UV Light
• There are three types of UV light o UV-A (320 – 400 nm) can cause some
DNA damage o UV-B (290 – 320 nm) is the major
lethal/mutagenic part of sunlight o UC-C (180 – 290 nm) is deadly
• The ozone layer absorbs all UV-C, some UV-B, and no UV-A.
Nucleic Acids 36
• A major mechanism of DNA damage by exposure to UV-B is the formation of thymine dimers. Here, two adjacent thymidines undergo a [2+2] photocycloaddition reaction that results in the formation of a cyclobutane ring.
N
HN
O
OR
CH3
N
NH
O
OR
H3C
N
HN
O
OR
N
NH
O
OR
UV
• The damage can enzymatically repaired, but excessive damages have been linked to skin cancer. Repair enzymes can excise the T-T dimers and replace them with normal T’s. Individuals that have a defective repair enzyme are more cancer-prone.
• With a normal ozone layer one hour of exposure to sunlight would generate 7 T-T dimers per 1000 reactive sites. If the ozone layer were halved, it would take 10 minutes. With no ozone, it would take 10 seconds.
• Sunscreens contain chromophores that absorb, as well as physical barriers (titanium dioxide) that shield, harmful UV light. (Look for those effective against UV-A and B).
Nucleic Acids 37
2. Carcinogens
• Up until the late 1970’s, benzene was liberally used in chemistry labs the same way we use acetone today: as a solvent for washing glassware and even hands. When studies linking benzene to leukemia and other cancers emerged around 1976, benzene usage was immediately restricted.
• The carcinogenicity of benzene is not due to benzene itself, but rather one of its metabolites, an epoxide.
OP450 DNA
OH
DNA
• Ironically, the body oxidizes benzene to the epoxide in an attempt to make more soluble for excretion. Toluene is a much safer alternative, because the benzyllic carbon oxidizes before the aromatic ring does.
COOH
Nucleic Acids 38
• Carcinogens are also made during home cooking. The burning of fat at high temp forms polyaromatic hydrocarbons (PAHs), such as benzo(α)pyrene.
• Its carcinogenicity is also aided by the fact that the molecule is large, planar, and hydrophobic. Thus, it can intercalate DNA and subsequently react with it.
HOOH
O DNA
HOOH
HO
• Carcinogens also formed by burning meat, which contains nitrogen from the amino acids. Toxic heterocyclic aromatic amines are formed, an example of which is shown.
N
N
N
NH2
Nucleic Acids 39
• Some carcinogens are natural products. In December 2005, many dogs died after eating food made by one manufacturer. The cause of death was liver failure due to aflatoxin, a deadly mycotoxin produced by Aspergillus flavus. (LD50 dog = 0.5 mg/kg)
O
O
O
H
H OCH3
O O
• The fungus can grow on corn, peanuts, and other grains, and the consumption of aflatoxin is a risk factor for human liver cancer (and death at higher doses).
• The underlying mechanism of toxicity is also caused by epoxidation of the alkene.
