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)

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

initiation

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

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')


A – U – G – U – U – U – G – U – C – A – G – U . . . . . mRNA (5')


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


mRNA (5')


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 . . . .



mRNA (5')


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 . . . .



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')


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

Shotgun Sequencing

Electrochemical Sequencing

http://www.youtube.com/watch?v=yVf2295JqUg

Chemistry 2100 Lecture 12. Purine/Pyrimidine Bases.

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Transcript of Chemistry 2100 Lecture 12. Purine/Pyrimidine Bases.