28.8 Nucleic Acids and Nucleotides - Vanderbilt...

15
212 418 Chapter 28 Biomolecules: Heterocycles and Nucleic Acids Heterocycles: cyclic organic compounds that contain rings atoms other than carbon (N,S,O are the most common). 28.1 Five-Membered Unsaturated Heterocycles (please read) 28.2 Structures of Pyrrole, Furan, and Thiophene (please read) 28.3 Electrophilic Substitution Reactions of Pyrrole, Furan, and Thiophene (please read) 28.4 Pyridine, a Six-Membered Heterocycle (please read) 28.5 Electrophilic Substitution of Pyridine (please read) 28.6 Nucleophilic Substitution of Pyridine (please read) 28.7 Fused-Ring Heterocycles (please read) These sections contain some important concepts that were covered previously. N H O S Pyrole Furan Thiophene Cyclopentadienyl anion 1 2 3 1 2 3 1 2 3 419 28.8 Nucleic Acids and Nucleotides Nucleic acids are the third class of biopolymers (polysaccharides and proteins being the others) Two major classes of nucleic acids deoxyribonucleic acid (DNA): carrier of genetic information ribonucleic acid (RNA): an intermediate in the expression of genetic information and other diverse roles The Central Dogma (F. Crick): DNA mRNA Protein (genome) (proteome) The monomeric units for nucleic acids are nucleotides Nucleotides are made up of three structural subunits 1. Sugar: ribose in RNA, 2-deoxyribose in DNA 2. Heterocyclic base 3. Phosphate

Transcript of 28.8 Nucleic Acids and Nucleotides - Vanderbilt...

212

418

Chapter 28 Biomolecules: Heterocycles and Nucleic Acids

Heterocycles: cyclic organic compounds that contain rings atoms other than carbon (N,S,O are the most common).

28.1 Five-Membered Unsaturated Heterocycles (please read)28.2 Structures of Pyrrole, Furan, and Thiophene (please read)

28.3 Electrophilic Substitution Reactions of Pyrrole, Furan, and Thiophene (please read)

28.4 Pyridine, a Six-Membered Heterocycle (please read)28.5 Electrophilic Substitution of Pyridine (please read)28.6 Nucleophilic Substitution of Pyridine (please read)28.7 Fused-Ring Heterocycles (please read)

These sections contain some important concepts that werecovered previously.

NH

O S

Pyrole Furan ThiopheneCyclopentadienyl

anion

1

2

3

1

2

3

1

2

3

419

28.8 Nucleic Acids and NucleotidesNucleic acids are the third class of biopolymers (polysaccharides

and proteins being the others)Two major classes of nucleic acids

deoxyribonucleic acid (DNA): carrier of genetic informationribonucleic acid (RNA): an intermediate in the expression

of genetic information and other diverse rolesThe Central Dogma (F. Crick):

DNA mRNA Protein (genome) (proteome)

The monomeric units for nucleic acids are nucleotidesNucleotides are made up of three structural subunits

1. Sugar: ribose in RNA, 2-deoxyribose in DNA2. Heterocyclic base3. Phosphate

213

420

Nucleoside, nucleotides and nucleic acids

The chemical linkage between monomer units in nucleic acidsis a phosphodiester

sugar base

sugar base

phosphate

sugar base

phosphate

sugar base

phosphate

sugar base

phosphate

nucleoside nucleotides

nucleic acids

421

The sugar: based on the furanose form D-riboseribose for RNA; 2-deoxyribose for DNA

OHOOH

HO OH

CHO

OHH

OHH

OHH

CH2OH

1

23

4

5

OHOOH

HO

D-ribose D-2-deoxyribose

The heterocyclic base; there are five common bases for nucleicacids

N

NH

N

N

N

N

purine

pyrimidine

N

NH

N

NN

NH

N

NH

NH2 O

NH2

NH

N

O

NH2

NH

NH

O

O

NH

NH

O

O

H3C

adenine (A)DNA/RNA

guanine (G)DNA/RNA

cytosine (C)DNA/RNA

thymine (T)DNA

uracil (U)RNA

1

2

34

5

67

8

9

1

2

3

4

5

6

214

422

Nucleosides: sugar + baseribonucleosides or 2’-deoxyribonucleosides

OHO N

HO

N

N

N

X

NH2

OHO N

HO

N

N

NH

X

O

NH2

RNA: X= OH, adenosine (A)DNA: X= H, 2'-deoxyadenosine (dA)

RNA: X= OH, guanosine (G)DNA: X= H, 2'-deoxyguanosine (dG)

OHO

HO X

N

N

O

NH2

OHO

HO

N

NH

O

O

H3C

RNA: X= OH, cytidine (C)DNA: X= H, 2'-deoxycytidine (dC)

DNA: thymidine (T)

OHO

HO

N

NH

O

O

RNA: R= H, uridine (U)

1

2

3

4

567

89

1'

2'3'

4'

5'

OH

423

Nucleotides: nucleoside + phosphate

28.9 Structure of nucleic acids: The chemical linkage between nucleotide units in nucleic acids is a phosphodiester, whichconnects the 5’-hydroxyl groupof one nucleotide to the3’-hydroxyl group of the next.

DNA is negatively charged

O

HO X

HO B

ribonucleoside (X=OH)deoxyribonucleoside (X=H)

ribonucleotide 5'-monophosphate (X=OH)deoxyribonucleotide 5'-monophosphate (X=H)

O

HO X

O BPHO

O

O

O

HO X

O BPO

O

O

PHO

O

O

O

HO X

O BPO

O

O

PO

O

O

PHO

O

O

nucleotide 5'-diphosphate nucleotide 5'-triphosphate

O

O OH

O B

P

O

O

ribonucleotide3',5'-cyclic phosphosphate

O

O

O X

Base

PO O

O

O

O X

Base

P

O

O O

3'

3'

5'

5'

5'

3'

215

424

DNA sequences are written from left to right from the 5’ to 3’

5’-ATCGCAT-3’ or 5’-d(ATCGCAT)-3’

28.10 Base Pairing in DNA: The Watson–Crick ModelChargaff’s Rule: the number of A = T and G = C in DNATwo polynucleotide strands, running in opposite directions

(anti-parallel) and coiled around each other in a double helix.

The strands are held together by complementary hydrogen- bonding between specific pairs of bases.

“Molecular Structure of Nucleic Acids” Watson J. D.; Crick, F. H. C. Nature 1953, 171, 737-738"Molecular Structure of Deoxypentose Nucleic Acids" Wilkins, M. H. F.; Stokes A.R.; Wilson, H. R.

Nature 1953,171, 738-740.”Molecular Configuration in Sodium Thymonucleate,” Franklin, R.; Gosling, R. G. Nature 1953,

171, 740-741

1962 Nobel Prize in Medicine: F. H. C. Crick, J. D. Watson, Maurice F. H. Wilkins,“for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.”

425

O

OR

RON

N

N

O

H

H

O

OR

RO

N

NN

NO

N

H

H

H

Hydrogen BondDonor

Hydrogen BondDonor

Hydrogen BondAcceptor

Hydrogen BondAcceptor

3'

5'

Hydrogen BondAcceptor

Hydrogen BondDonor

5'

O2

N4 O6

3'

Antiparallel C-G Pair

N3 N1

N2

O

OR

RON

N

O

O

O

OR

RO

N

N N

NN

H

H

H

5'

3'

3'

5'

Hydrogen BondDonor

N6

Hydrogen BondAcceptor

Hydrogen BondAcceptor O4

Hydrogen BondDonor

Antiparallel T-A Pair

N1N3

Anti-parallel, complementary hydrogen-bonding of DNA base pairs

216

426

DNA double helix

one helical

turn34 Å

major groove12 Å

minor groove

6 Å

backbone: deoxyribose and phosphodiester linkagebases

427

DNA Grooves

cytidine guanosine

thymidine adenosine

minor groove

major groove

minor groove

major groove

217

428

28.11 Nucleic Acids and HeredityThe Central Dogma (F. Crick):

DNA replication DNA transcription mRNA translation Protein (genome) (proteome)

Expression and transfer of genetic information:Replication: process by which DNA is copied with very

high fidelity. Transcription: process by which the DNA genetic code

is read and transferred to messenger RNA (mRNA).This is an intermediate step in protein expression

Translation: The process by which the genetic code isconverted to a protein, the end product of gene expression.

The DNA sequence codes for the mRNA sequence, whichcodes for the protein sequence

“It has not escaped our attention that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

429

28.12 Replication of DNA: DNA is replicated by the coordinatedefforts of a number of proteins and enzymes.

Each cell contains about two meters of DNA. The DNA mustbe “packaged” into the cell nucleus by super-coiling and knotting.

For replication, DNA must be unknotted, uncoiled and thedouble helix unwound.

Topoisomerase: Enzyme that unknots and uncoils DNAHelicase: Protein that unwinds the DNA double helix. DNA polymerase: Enzyme replicates DNA using each strand as

a template for the newly synthesized strand.

DNA replication is semi-conservative: Each new strand of DNAcontains one parental (old, template) strand and onedaughter (newly synthesized) strand

218

430

Unwinding of DNA by helicases expose the DNA bases (replication fork) so that replication can take place

leading strandDNA polymerase δ

Lagging strandDNA polymerase ε

DNA replication

431

DNA Polymerase: the new strand is replicated from the 5’ → 3’(start from the 3’-end of the template) DNA polymerases are Mg2+ ion dependentThe deoxynucleotide 5’-triphosphate (dNTP) is the reagent

for nucleotide incorporation

G

O

O

OP

O

-O

A

O

O

O

T

O

OH

O P O

O

C

O

OH

O

O-PO-

O

O P

O-

O

O-

Mg2+

5'

3'

5'

templatestrand(old)

(new)

3’-hydroxyl group of the growing DNA strand acts as a nucleophile and attacks the α-phosphorus atom of the dNTP.

dNTP

219

432

Replication of the leading strand occurs continuously in the 5’ → 3’ direction of the new strand.

Replication of the lagging strand occurs discontinuously. ShortDNA fragments are initially synthesized and then ligated together. DNA ligase catalyzes the formation of the phosphodiester bond between pieces of DNA.

animations of DNA processing: http://www.wehi.edu.au/education/wehi-tv/dna/

433

DNA replication occurs with very high fidelity:Most DNA polymerases have high intrinsic fidelityMany DNA polymerases have “proof-reading”

(exonuclease) activityMismatch repair proteins seek out and repair base-pair

mismatches due to unfaithful replication

28.13 Structure and Synthesis of RNA: TranscriptionRNA contains ribose rather than 2-deoxyribose and uracil rather

than thymineThere are three major kinds of RNA

messenger RNA (mRNA): ribosomal RNA (rRNA)transfer RNA (tRNA)

DNA is found in the cell nucleus and mitochondria; RNA is moredisperse in the cell.

220

434

Transcription: only one of the DNA strands is copied (codingor antisense strand). It sequence is converted to thecomplementary sequence in mRNA (template or sense strand), which codes for the amino acid sequence of a protein (or peptide)

28.14 RNA and Protein Biosynthesis: Translation• proteins are synthesized in the cytoplasm on ribosomes.• mRNA is the template for protein biosynthesis.• a three base segment of mRNA (codon) codes for a

an amino acid.

435

The “anticodon” region of of tRNA is complementary for ato the mRNA codon sequence.

The t-RNA carries an amino acid on the 3’-hydroxyl (aminoacylt-RNA) and the ribosome catalyzes amide bond formation.

Although single-stranded, the are complementary sequences within tRNA that give it a defined conformation

aminoacyl t-RNA

221

436

U G U AA U C U CG U U

3'

A CU

OO

H2N

H3CS

5'

U G U AA U C U CG U U

3'

A CU

OO

H2N

H3CS

5'

U AA

OO

H2N

OH

U G U AA U C U CG U U

3'

A CU

5'

U AA

OO

HN

OHOH2N

H3CS

HO

U G U AA U C U CG U U

3'

A CU

5'

U AA

OO

HN

OHOH2N

H3CS

HO

U G U AA U C U CG U U

3'5'

U AA

OO

HN

OHOH2N

H3CS

G AC

O O

CH3H2N

Ribosomal protein synthesis

437

DNA sequencingMaxam-Gilbert: relys on reagents that react with a specific

DNA base that can subsequent give rise to asequence specific cleavage of DNA

Sanger: Enzymatic replication of the DNA fragment to be sequenced with DNA polymerase, Mg+2, and dideoxynucleotides triphosphate (ddNTP) that truncate DNA replication

Restriction endonucleases: Bacterial enzymes that cleaveDNA at specific sequences

5’-d(G-A-A-T-T-C)-3’3’-d(C-T-T-A-A-G)-5’

5’-d(G-G-A-T-C-C)-3’3’-d(C-C-T-A-G-G)-5’

EcoR I

BAM HI

5'

3'

3'

5'

5'

3'

3'

5'

OH 2-O3PO

2-O3PO OH

restrictionenzyme

222

438

Sanger Sequencing dideoxynucleotides triphosphate (ddNTP)

N

NN

N

NH2

OOPO

O-

O

P

O

O-

OP

O

-O

O-

NH

NN

N

O

OOPO

O-

O

P

O

O-

OP

O

-O

O-

ddATP

NH2

ddGTP

OOPO

O-

O

P

O

O-

OP

O

-O

O-

OOPO

O-

O

P

O

O-

OP

O

-O

O-

ddTTP ddCTP

N

NH

O

ON

N

NH2

O

When a ddNTPis incorporatedelongation of the primer is terminated

The ddNTP isspecificallyincorporatedopposite itscomplementarynucleotide base

DNA polymerase

3'

5'-32P3'

5'

primer

Template

unknown sequence

Mg2+, dNTP's

+ one ddATP

O

N3

HO N

NH

O

OOHO N

N

N

NH

O

S

O OHN

N

H2N

OOHO N

N

NH2

O

ddIAZT

(-)-3TC

Anti-Viral Nucleosides

d4C

439

Sanger Sequencing

Larger fragments

Smallerfragments

5'

3'

32P-5' 3'

primer template

G

T

A

A

C

G

T

A

A

T

C

A

C

A

G

ddA ddG ddC ddT

C

A

T

T

G

C

A

T

T

A

G

T

G

T

C

5'

32P

3'

223

440

Automated Sanger Sequencing with flourescent ddNTP's

Excitation: ~ 490 nM, Emission: ddT= 526 nm. ddC= 519 nm, ddA= 512 nm, ddG= 505 nm

N

NN

NH2

O

-3 H3O9P3O

HN

ON

CH3

O

OO O -

CH3H3C

NH

NN

O

O

-3 H3O9P3O

HN

ON

CH3

O

OO O -

NH2

OO O -

O

-3 H3O9P3O

N

N

NH2

O

HN O

NH3C

(CH2)2O

CH3CH3

OO O -

CH3H3CO

-3 H3O9P3O

HN

N

O

O

HN O

NH3C

(CH2)2O

CH3CH3

ddA ddGddT ddC

Sanger sequencing using flourescent ddNTP's: terminated DNA strands are separated by capillary electrophoresis, detected and identified by their fluorescence emission

441

Sequencing on a “chip” (Lagniappe)Spatially addressable: 8-mer chip: 65, 536 different sequences

12-mer chip: 1,677,216 different sequences

DNA fragment DNA–fluorophore

Place DNA on the chip, then wash awaynon-specific hybridization after 1-10 hrs. Raise temperature and “melt” awaypartially hybridized sequences.

3’-ACGGTGCG CGGTGCGA

GGTGCGAG GTGCGAGA TGCGAGAA GCGAGAAT etc

3’-ACGGTGCGAGAAT---5’ (from the chip)5’-TGCCACGCTCTTA---3’

224

442

28.16 DNA Synthesis: short segments of DNA can be efficiently by automated, solid-phase methods.

Solid support: controlled pore glass (silica)linked to the 3’-hydroxyl group of the first nucleotidesolid phase DNA synthesis is from 3’→5’, which is theopposite direction from nature.

Protected nucleotide reagents: 5’-protecting group: 4,4’-dimethyoxytrityl (trityl is a triphenylmethyl group), abbreviated DMT

5’-DMT ether C O

OCH3

OCH3

O

HO

Base

Removes with mild acid(Cl3CCO2H)

443

The base amino groups of dA, dG and dC must be protected,usually as an amides. The base of T does not require further protection.

N

N N

NN

N N

NH

HN O

NH

N

N

O

HN

N

NH

O

O

H3C

G

R R R R

O

O

O

TCA

The 3’-phosphorous group: phosphoramidite

OHO B

HO

Base is protected

DMTr-Cl

pyridine

ODMTrO B

HO

NPO

Cl

CN

pyridine

ODMTrO B

O

PO N[CH(CH3)2]

NC

225

444

OH O

O

O

Si (CH2)3 NH2

O

O

O

Si (CH2)3 NH

C (CH2)2

O

C

O

OH

O

O

O

Si (CH2)3 NH

C (CH2)2

O

C

O

O

B1

O

DMT-O

S

O

B

O

PO

O

O

B1

O

DMT-O

CN

PN (iPr)2

O

O

B2

O

DMT-O

CN

OH

OH

O

B1

O

HO

SILICA

SILICA

SILICA

SILICA

Cl3CCOOH

S

Cl3CCOOH

NH3, H2O, !

I2, H2O

coupling

N

NN

NH

oxidation of P

5'- deprotection

O

B1

O

P

O

O

O

B2

O

DMT-O

OCN

S

Cl3CCOOH

5'- deprotection

repeat cycle

O

B1

O

P

O

O

O

B2

O

O

OCN

S

P

O

O

B3

O

DMT-O

OCN

O

B1

O

P

O

O

O

B2

O

O

OCN

S

P

O

O

B3

O

HO

OCN

5'- deprotection

O

B1

OH

P

O

O

O

B2

O

O

O

P

O

O

B3

O

HO

Odeprotect Pand the baseamino groups

Automated, solid-phase DNA synthesis

445

28.17 Polymerase Chain Reaction (PCR): method foramplifying DNA using DNA polymerase and cycling temperature

Heat stable DNA Polymerases (from archaea):Taq: thermophilic bacteria (hot springs)- no proof readingPfu: geothermic vent bacteria- proof reading

Mg 2+

two Primer DNA strands (synthetic, large excess)one sense primer and one antisense primer

one Template DNA stranddNTP’s O B

HO

OPO

O-

O

P

O

O

O-

P-O

O-

O

KARY B. MULLIS, 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction (PCR) method.

226

446

A typical PCR temperature cycle

Denaturation: 94 °C 0.5 - 1 min

Annealing: 55-68 °C 0.5 - 1 min 5 °C below the Tm of the primer

Extension: 72 °C 1 min + 1 min per Kb (replication) of DNA

# of cycles 25 - 35

Final extension 72 °C 10 min

1 x 2 = 2 x 2 = 4 x 2 = 8 x 2 = 16 x 2 = 32 x 2 = 64 x 2 = 128 x 2 = 256 x 2 = 512 x 2 = 1,024 x 2 = 2,048 x 2 = 4,096 x 2 = 8,192 x 2 = 16,384 x 2 = 32,768 x 2 = 65,536 x 2 = 131,072 x 2 = 262,144 x 2 = 524,288 x 2 = 1,048,576

In principle, over one million copies per original, can be obtained after just twenty cycles

447

Polymerase Chain Reaction

For a PCR animation go to: http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/pcr.html http://users.ugent.be/~avierstr/principles/pcrani.html

5'

3'

3'

5'

95 °C

denaturation

5' 3'

3' 5'

anneal (+) and (-) primers

55 - 68 °C

5' 3'

3' 5'

3'

3'

72 °C

Taq, Mg 2+, dNTPs

extension

5'

3'

3'

5'

5'

3'

3'

5'

95 °C

denaturation

2nd cycle

5'

3'

3'

5'

5'

3'

3'

5'

amplification of DNA

2 copies of DNA

repeat temperature cycles