Chemistry 2100 Lecture 12. Purine/Pyrimidine Bases.
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Transcript of Chemistry 2100 Lecture 12. Purine/Pyrimidine Bases.
Chemistry 2100
Lecture 12
Purine/Pyrimidine Bases
HN
NO
H
N
N
NH2
H
HN
N
H
CH3
Uracil (U)(in RNA only)
Thymine (T)(DNA only)
Cytosine (C)(DNA andsome RNA)
N
N
Pyrimidine
1
2
3
4
5
6
HN
N N
NO
HH2N
Guanine (G)(DNA and RNA)
N
N N
N
NH2
HAdenine (A)
(DNA and RNA)
N
N N
N
HPurine
1
2
3
4
56 7
8
9
O O
O O
-N-glycoside
adenosine[a nucleoside]
adenosine monophosphate[a nucleotide]
1'2'3'
5'
4'
adenine
ribose
9
P OHHO
OH
O
N
N
N
N
NH2
H
O
N
N
N
N
NH2
CH2
HO OH
HOOH
OHHO
CH2O
HO
(-H 2O)
(-H 2O)O
N
N
N
N
NH2
CH2
HO OH
OP
O
HO
OH
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(- H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(- H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(- H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(- H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(- H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(-H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(-H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(-H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(-H2O)
H
OH
O
O
P O CH2
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
OH
H
H
N
N
NH2
O
H
H
OH
O
O
P O
O
CH2O
N
N
O
O
HCH3
H
OH
O
O
P O
O
CH2O
N
N
N
N
O
NH2
H
OCH2
O
OP
O
O
O
OCH2
O
OPO
O
O
NH2
N
N
OCH2
O
OPO
O
CH2OPO
O
CH3 H
O
O
N
N
(etc.)
(etc.)
3'
5'
(etc.)
(etc.)
(-H2O)
S TP
S GP
S CP
P
(etc.)
(etc.)
Hydrogen Bonding!
Discovery of DNA Structure
• One of the most important discoveries in biology
• Why is this important– "This structure has novel features which are of
considerable biological interest“--- Watson and Crick, Nature, 1953
• Good illustration of science in action:– Missteps in the path to a discovery– Value of knowledge– Value of collaboration– Cost of sharing your data too early
Covalent Structure of DNA (1868-1935)
• Friedrich Miescher isolates “nuclein” from cell nuclei
• Hydrolysis of nuclein:– phosphate– pentose– and a nucleobase
• Chemical analysis:– phosphodiester linkages– pentose is ribofuranoside
O
OH
H HH
Thymine
H
CH2O P
OH
O
O
P OOH
OH
O
H
H HH
Adenine
H
CH2O P
OH
O
O
Structure of DNA: 1929
(Levene and London)
Structure of DNA:
1935(Levene and Tipson)
C5H7OThymine
O
P
O
POH
OH
O
O
O
OH
C5H7OAdenine
O
Road to the Double Helix• Franklin and Wilkins:
–“Cross” means helix
–“Diamonds” mean
that the phosphate-
sugar backbone
is outside
– Calculated helical
parameters
• Watson and Crick:
– Missing layer means
alternating pattern
(major & minor groove)
– Hydrogen bonding:
A pairs with T
G pairs with C
Double helix fits the data!
Watson, Crick, and Wilkins shared 1962 Nobel Prize
Franklin died in 1958
Other forms of DNA
DNA Replication
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”
Watson and Crick, in their Nature paper,1953
Step 1: helicase
Step 2: DNA polymerase
Step 3: DNA ligase
Why detect Transcription Factor targets?
• Transcription factors are medically relevant– ~10% of human genes– Crucial roles in
development and cell life cycle
– Misregulation and mutation cause disease
– Critically, most cancers involve TF overactivity
Darnell, Nature Reviews Cancer 2, 740 (2002)
Traditional methods for Transcription Factor
detectionExpression Microarrays Gel Shift Assays
The challenge: Most of these methods are indirect, slow
(hours), or can’t differentiate active and inactive protein.
Western Blots
Bio-mimicry is a powerful motivation
Velcro: inspired by burrs Conformation Switching Probes
Marvin J S et al. PNAS 1997;94:4366-4371
Optical Conformation SwitchingTF Switch Sensors
Rationally Tuning TF Sensors
% s
wit
ches
open
KS = 10
KS = 1
KS = 0.1
KS
= 0
.01
KS
= 0
.001
KS = [ ]
[ ]
KS [target]
KD (1+ KS) + KS [target] % switches open =
KD = [ ]
[ ]
[ ]
Target [M]
TF Beacon Actual Performance
Quantitative Detection in 4 easy steps
transcription
transcription
transcription
initiation
initiation
initiation
elongation
elongation
elongation
elongation
elongation
elongation
elongation
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA
tRNA
peptide Met –––– Phe –––– Val –––– Ser . . . .
(5')
U – A – C – A – A – A – C – A – G – U – C – A
AU
C U
HONO
N
N
NH2
O
H
H2O
N
NO
H
OH
N
N
H
O
O
H
Pyrimidine Dimers from UV
http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
Val
H2N H
COOH
CH(CH3)2
GUAGUG
Glu
H2N H
COOH
CH2CH2COOH
GAAGAG
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
Val
H2N H
COOH
CH(CH3)2
GUAGUG
Glu
H2N H
COOH
CH2CH2COOH
GAAGAG
ACUACCACAACG H OH
CH2OH
COOH
HH2N
Thr
UCUUCCUCAUCG
H2N H
COOH
CH2OHSer
Val
H2N H
COOH
CH(CH3)2
GUAGUG
Glu
H2N H
COOH
CH2CH2COOH
GAAGAG
DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
DNAT – A – C – A – A – A – C – A – G – T – C – A . . . . .
A – T – G – T – T – T – G – T – C – A – G – T . . . . .
(3')
(5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')
peptide Met –––– Phe –––– Asp –––– Ser . . . .
A – U – G – U – U – U – G – A – C – A – G – U . . . . . mRNA (5')
Mutations
point substitution mutation
peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
peptide Met –––– Phe –––– Asp –––– Ser . . . .
A – U – G – U – U – U – G – A – C – A – G – U . . . . . mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
point substitution mutation
peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
peptide Met –––– Phe –––– Asp –––– Ser . . . .
A – U – G – U – U – U – G –A– C – A – G – U – A . . . . mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
point substitution mutation
peptide Met –––– Phe –––– Val –––– Ser . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . . mRNA (5')
peptide Met –––– Phe ––– Asp ––– Ser . . . .
A – U – G – U – U – U – G –A– C – A – G – U – A . . . . mRNA (5')
peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
frameshift mutation
peptide Met –––– Phe –––– Val –––– Val . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
frameshift mutation
peptide Met –––– Phe –––– Val –––– Val . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
peptide Met –––– Phe –––– Val –––– Ser . . . .
mRNA (5')
frameshift mutation
peptide Met –––– Phe –––– Val –––– Val . . . .
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .mRNA (5')
A – U – G – U – U – U – G – U – C – A – G – U – A . . . .
PCR
PolymeraseChainReaction
DNA Fingerprinting
DNA Sequencing
DNA Sequencing
Shotgun Sequencing
Electrochemical Sequencing
http://www.youtube.com/watch?v=yVf2295JqUg