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9/30/14 Nucleic Acids
Learning objectives
1) 
2) 
3) 
4) 
5) 
6) 
Iden-fy building blocks, purine and pyrimidines, of nucleics acids. Draw base versus nucleoside versus nucleo-de structures. Compare chemical structures of ribonucleoside versus deoxyribonucleoside. Diagram forma-on of a phosphodiester bond; how is this structure stabilized? Explain the structural basis for Chargaff’s rules. Diagram double stranded helix of DNA including 5’ and 3’ strand, sugar phosphate backbone, major and minor groves, and hydrogen bonding paRern between strands. 7)  List the structural differences between DNA and RNA. 8)  Explain why the double-­‐stranded nature of DNA is relevant for copying and transmiXng gene-c informa-on when a cell divides. 9)  Summarize the steps required to amplify a given segment of DNA in vivo and in vitro. 10) Explain the role of DNA muta-ons in evolu-on. 11) Explain how nucleic acid thermodynamics are u-lized in molecular biology techniques to characterize and manipulate DNA. 12) Describe the ac-vi-es of the enzymes required to construct a recombinant DNA molecule. 13) Explain process of sequencing DNA using dideoxy DNA sequencing method. 14) Summarize what is known about the size and gene content of the human genome. Chemistry & Biochemistry – F2014
Nucleic acids - components
Chemistry & Biochemistry – F2014
Nucleic acids - components
Sugar
1
① 
② 
③ 
④ 
Nucleic acids form the largest polymer in the cell.
Individual unit of the polymer is called a nucleotide or nucleic acid.
Individual units are linked together via a condensation reaction.
The individual unit is composed of phosphate, sugar, heterocyclic base.
Relative abundance in solution
2
3
4
59%
20%
13%
7%
5
0.1%
Phosphate group
Involved in ring
closure
Used to bond to
heterocyclic base
Bonds phosphate unit
of this nucleotide
Phosphoanhydride
bond
PPi + H2O à 2Pi
ΔG (kJ/mol) = -31.8
3’OH 2’OH
①  Polyanionic
②  Resonance stabilization
③  Linking phosphoric acid
through anhydride linkages
to store energy
④  Large –ve ΔG from
resonance stabilization and
solvation of product
Chemistry & Biochemistry – F2014
Helpful mnemonic (maybe):
Nucleoside = has only a
nitrogenous base joined to
one side
Nucleic acids - components
Heterocyclic bases
Nucleoside Nomenclature
-  add suffix ‘-idine’ to pyrimidine
-  add “-osine” to purine
-  Pyrimidine nucleosides:
-  Cytidine, Thymidine, Uridine
-  Purine nucleosides:
-  Adenosine, Guanosine
Used to bond to
phosphate group of other
nucleotides
Distinguish between RNA
ribose & DNA
deoxyribose
To initiate nucleotide synthesis, the ribose is phosphorylated at the 5’OH
ATP + Ribose ßà ADP + Ribose-5-phosphate
Chemistry & Biochemistry – F2014
Nucleic acids – connecting components
pyrimidine synthesis
+
Linkage between
ribose and base is
an N-link
glycosidic bond
Salvage pathway
recovers base &
adds to R-5-P
Salvage pathway
has nucleoside
intermediate
purine synthesis
uracil (U)
guanine
adenine
cytosine
thymine
Orotate
uracil
5-phosphoribulose-1diphosphate
guanosine
adenosine
cytidine
thymidine
uridine
Chemistry & Biochemistry – F2014
Helpful mnemonic (maybe):
Nucleotides = has two groups joined to the sugar.
Nucleoside = has only a nitrogenous base joined to one side
UTP
Chemistry & Biochemistry – F2014
1 9/30/14 Nucleic acids – nucleotides
Nucleic acids – connecting components
Formation of a phosphodiester bond
NAD
Coenzyme A
FAD
Role of nucleotides:
①  Building blocks of RNA and DNA
②  Storage unit of energy
③  Cofactors
④  Signaling
AMP
ΔG ≅ -50 kJ/mol
GTP
Base
Nucleoside
Nucleotide
DNA
Adenine (A)
Guanine (G)
Cytosine (C)
Thymine (T)
deoxyadenosine dAMP dADP dATP
deoxyguanosine dGMP dGDP dGTP
deoxycytidine dCMP dCDP dCTP
deoxythymidine dTMP dTDP dTTP
RNA
Adenine (A)
Guanine (G)
Cytosine (C)
Uracil (U)
deoxyadenosine
deoxyguanosine
deoxycytidine
deoxyuridine
AMP
GMP
CMP
UMP
ADP
GDP
CDP
UDP
ATP
GTP
CTP
UTP
Cyclic AMP
Chemistry & Biochemistry – F2014
Nucleic acids – RNA vs DNA structure
What it is: 5’-GCAT-3’
What is written: GCAT
What it is: 5’-TACG-3’
What is written: TACG
Chemistry & Biochemistry – F2014
Nucleic acids – History of defining the DNA duplex
Chargraff’s rule
Tautomer
Jerry Donahue role in determining the structure of DNA…
Donohue was born in Sheboygan, Wisconsin.
Dartmouth: AB and MA
Caltech: PhD under Linus Pauling
Donohue remained at Caltech until 1952.
In their famous article by Watson and Crick in Nature that proposed the
structure of DNA, the following acknowledgment to Donohue appears:
"We are much indebted to Dr. Jerry Donohue for constant advice and
criticism, especially on interatomic distances".
Chemistry & Biochemistry – F2014
Nucleic acids – History of defining the DNA duplex
Chemistry & Biochemistry – F2014
Nucleic acids – base pairing
Conformation of base
Base pairing constrained by duplex diameter & H-bond pattern
Normal: Watson-Crick pairing
H-bonds are 180°, short (strong)
Alternate H bonds: Hoogsteen pairing
Requires syn conformation
H-bonds are not 180°, GC reduced from 3 to 2 H bonds
Chemistry & Biochemistry – F2014
Chemistry & Biochemistry – F2014
2 9/30/14 Nucleic acids – base pairing
Nucleic acids – History of defining the DNA duplex
Diffraction pattern of vertically oriented DNA fiber
10
9
8
7
6
5
4
3
The only other purine-pyrimidine pairings would be AC and GT and UA (in RNA).
2
1
0
Ex. Wobble base pair
A
C
G
T
A
P
Layer lines
between 0 and
strong meridian
reflection
indicate repeat
(ie turn) of
helical structure
Pitch; spacing
between
repeating bases
U
Pairings are mismatches because the pattern of hydrogen donors and
acceptors do not correspond
Wobble hypothesis: Crick postulated that the 5' base on the
anticodon, which binds to the 3' base on the mRNA, was not as
spatially confined as the other two bases, and could, thus, have nonstandard base pairing
A wobble base pair is a pairing
between two nucleotides in RNA
molecules. The thermodynamic
stability of a wobble base pair is
comparable to that of a Watson-Crick
base pair. Wobble base pairs are
fundamental in RNA secondary
structure and translation.
Strong meridian
reflection
X pattern
indicative of
helical structure
Chemistry & Biochemistry – F2014
Nucleic acids – DNA duplex forms
Chemistry & Biochemistry – F2014
Nucleic acids – Other base paired structures
Physiological examples
A-form: DNA-RNA hybrid;
double-stranded RNA's
B-form: DNA duplex
Z-DNA: alternating purinepyrimidine sequence (especially
poly(dGC)2); negative DNA
supercoiling or high salt and
some cations; structure which
involves the extrusion of a base
pair; Does not exist as a stable
feature of the double helix.
Geometry attribute
A-­‐form
Handedness right
repeating unit 1 bp
rotation/bp 33.6°
mean bp/turn
11
Rise/turn of helix 24.6Å
Glycosyl angle
Diameter
anti
23Å
B-­‐form
right
1 bp
35.9°
10.5
33.2Å
anti
20Å
Z-­‐form
left
2 bp
60°/2
12
45.6Å
Pyr: anti; pur: syn
18Å
Chemistry & Biochemistry – F2014
Nucleic acids – History of defining the DNA duplex
"It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic material."
With this modest understatement, Watson and Crick introduced the second major
discovery brought about by an understanding of DNA's structure, the mechanism by
which DNA makes copies of, or replicates , itself. Finally, a mechanism for
understanding how traits are passed on from parent to child was apparent.
Chemistry & Biochemistry – F2014
Nucleic acids – base pairing
①  The regular structure and data
redundancy provided by the DNA
double helix makes DNA well suited to
the storage of genetic information.
②  The base-pairing between DNA and
incoming nucleotides provides the
mechanism through which DNA
polymerase replicates DNA.
③  The base-pairing of RNA to DNA allows
RNA polymerase to synthesize mRNA.
④  DNA-binding proteins can recognize
specific base pairing patterns that
identify particular regulatory regions of
genes.
Chemistry & Biochemistry – F2014
Chemistry & Biochemistry – F2014
3 9/30/14 Nucleic acids – DNA sequence conventions
1)  In a DNA duplex, strands are
anti-parallel.
2)  Individual strands are
synthesized from the 5’ OH of
the ribose to the free 3’OH at the
terminus of the strand. Thus,
DNA sequences have
directionality going from 5’ to 3’.
3)  The anti-parallel strands are said
to be complimentary due to the
hydrogen bonding occurring
between heterocyclic bases
across strands of duplex.
4)  DNA sequences are written 5’ to
3’
5)  When a complimentary strand is
given for the 5’ to 3’ strand, this
is the reverse complement as it
is the 3’ strand sequence
rewritten from 5’ to 3’
DNA replication
When/Why is DNA replicated?
If sequence 5’-TGC-3’, then what is the
complement?
a)  3’-TGC-5’
b)  3’-ACG-5’
c)  3’-GCA-5’
d)  3’-CGT-5’
How does DNA duplex separate?
How is synthesis of new DNA initiated?
The strands are running opposite directions. How does this
affect synthesis of the two strands?
If sequence 5’-ATCCG-3’, then what is
the complement?
a)  5’-GCCAT-3’
b)  5’-GCCTA-3’
c)  5’-TAGGC-3’
d)  5’-CGGAT-3’
If given sequence GATTACAC, what is
complement?
Chemistry & Biochemistry – F2014
Chemistry & Biochemistry – F2014
DNA replication – Steps in the process
DNA replication – Steps in the process
1
3
1)  Breaking open the DNA duplex
a) A-T rich regions
b) DNA gyrase (enzyme) nicks DNA strand
c)  Helicase (enzyme) splits two strands
d) Each strand serves as a template (semi-conservative
replication)
e) Initiation point = “origin of replication”
f)  structure formed where DNA opens = “Replication Fork”
2
2)  Priming the system
a) RNA Primase (enzyme) binds in the initiation point of
the 3'-5' parent chain.
b)  RNA Primase can attract RNA nucleotides which bind
to the DNA nucleotides of the 3'-5' strand due to the
hydrogen bonds between the bases.
c)  RNA nucleotides are the primers (starters) for the
binding of DNA nucleotides.
Chemistry & Biochemistry – F2014
DNA replication – Steps in the process
4
3)  Elongation Process
a) DNA polymerase III (DNA Pol III – enzyme)
synthesizes complementary strand
b) 5’ - 3’ template
i.  Leading stranding
ii.  Continuous addition of nucleotides
c) 3’ – 5’ template
i.  Lagging strand
ii.  Additional RNA primers required
iii. Replicated segments = Ozaki fragments
4)  Sewing together the pieces
a) DNA Pol I examines Ozaki fragments and removes
RNA primers
b) DNA Pol III fills gaps with complementary nte.
c) DNA ligase (enzyme) connects fragments by
completing phosphodiester linkages.
Chemistry & Biochemistry – F2014
Complimentarity of DNA/RNA in transcription & translation
5
5)  Termination
a) DNA polymerase III reaches end of strand
b) Lagging strand cannot replicate to end of strand
6
6)  Repair
a) Nucleases excise any errors in replicating DNA
(mismatches)
b) DNA Pol III fills gaps with complementary nte.
Chemistry & Biochemistry – F2014
Transcription: RNA polymerase
synthesizes a messenger RNA (mRNA)
sequence that is complimentary to DNA
template.
Translation: tRNA anticodons
compliment mRNA codons to deliver
amino acids to growing polypeptide
chain.
Chemistry & Biochemistry – F2014
4 9/30/14 Deciphering the code
How many bases would be in each code word, or codon?
What was known:
1)  4 bases; 20 amino acids
a)  George Gamow reasoned:
i.  1 bp / codon = 4
ii.  2 bp / codon = 16
iii.  3 bp/ codon = 64
2)  Genes were defined entity & linear
a)  Seymour Benzer using phage mutations showed:
i.  Linearly structured map of a genetic region
ii.  Resolved mutants to single nte Δes
3)  Triplets are degenerate:
a)  Crick & Brenner found:
i.  Using Benzer’s phage approach, confirmed triplet
nature of genetic code
ii. Using frameshift mutations found that when 3 bp were
added to or deleted from a gene, the encoded protein
was minimally affected
iii. Codons were non-overlapping
iv. Defined starting point
4)  Determining the cipher:
a)  Nirenberg & Matthaei
i.  Cell-free system that adds amino acids into
polypeptide chain
ii. Required addition of mRNA – could be defined
iii. Used single polynucleotide
Deciphering the code
E. coli extract
(tRNAs, ribosomes,
ATP, etc.)
mRNA-UUUUUUUUUUUUUU
A C D E F G H I K L
M N P Q R S T V W Y
Codon table:
64 possibilities
3 stop codons
The “wobble” at position 3.
Chemistry & Biochemistry – F2014
Chemistry & Biochemistry – F2014
The prose that begets a protein
Relationship between macromolecular building blocks
Start codon – sequence – Stop codon
Central dogma of molecular biology
ATG-AGT-GGG-CAT-AAA-CGT- XXXN –CCC-GAA-GAA-AUU-ACC-UGU-TAA
M
S
G
H
K
R
X
P
E
E
I
T
C
*
Flow of genetic information in a biological system
prions
RNA viruses
Reverse transcriptase
Retroviruses
Chemistry & Biochemistry – F2014
Nucleic acids – Thermodynamics
Chemistry & Biochemistry – F2014
ΔrG = -RTlnKeq
Nucleic acids – Thermodynamics
van’t Hoff eq
Ass + Bss ßà Cds
1)  Thermal denaturation = melting
2)  Denaturation occurs through disruption of
hydrophobic stacking of bases and
hydrogen bonding between bases
3)  Melting temperature (Tm) defined as the
temperature at which half of the DNA
strands are in the random coil or singlestranded (ssDNA) state.
4)  Tm depends on the length and sequence.
a)  hGC content = higher Tm
b)  Mismatches = iTm
5)  Hybridization = process of establishing a
non-covalent, sequence-specific
interaction between two or more
complementary strands of nucleic acids
into a single complex
6)  Annealing = pairing of complementary
sequences via hydrogen bonding of
bases – usual refers to short sequences,
ie primers, used in in vitro rxns.
Keq =
[Cds]
[Ass][Bss]
α=
2[Cds]
CT
[Bss] =
Keq =
=
CT - 2 [Bss]
CT
(1 – α)CT
αCT/2
[(1-α)CT/2]2
Keq =
=1 - 2
[Cds] =
2
=
4
CT
-lnKeq = (ΔrH/RT) – (ΔrS/R)
Eq constant for
single to duplex
formation
CT = [Ass] + [Bss] + 2[Cds]
= 2[Bss] + 2[Cds]
ΔrG = ΔrH - TΔrS
1/T = -(R/ΔrH)lnKeq + (ΔrS/ΔrH)
Define total
strand
concentration
Fraction of total
DNA in duplex
form
[Bss]
CT
αCT
2
2α
(1-α)2CT
Define ss and ds
concentrations
DNA denaturation
is reversible
Substitute &
cancel
When 50% strand
melted (Tm), then α
= 1/2
1/Tm = -(R/ΔrH)ln4/CT+ (ΔrS/ΔrH)
Chemistry & Biochemistry – F2014
y = m*x + b
van’t Hoff plot
Chemistry & Biochemistry – F2014
5 9/30/14 Nucleic acids – Thermodynamics
Nucleic acids – Manipulation
Polymerase Chain Reaction (PCR)
Example of van’t Hoff plot of DNA melting data
1/Tm = -(R/ΔrH)ln4/CT+ (ΔrS/ΔrH)
Amplifies DNA based upon primer set.
Used in cloning, DNA sequencing, DNA fingerprinting & diagnosis of hereditary
disease, infections…
Manipulate DNA sequence by including additional sequences in primer sequences.
y = m*x + b
Chemistry & Biochemistry – F2014
Mutating DNA sequences
Chemistry & Biochemistry – F2014
Cloning DNA sequences
Ethidium Bromide
Repurposing bacterial defenses for biotech
Chemistry & Biochemistry – F2014
Genetic engineering: Producing recombinant proteins
Chemistry & Biochemistry – F2014
Chemistry & Biochemistry – F2014
DNA – Reading the sequence
Chemistry & Biochemistry – F2014
6 9/30/14 Basis for DNA sequencing
DNA sequencing
Formation of a phosphodiester bond
Chemistry & Biochemistry – F2014
DNA sequencing approaches
Chemistry & Biochemistry – F2014
Genomic sequencing
Shotgun sequence approach
Chemistry & Biochemistry – F2014
Define differences in DNA sequences: Fingerprinting
Chemistry & Biochemistry – F2014
Define differences in DNA sequences: Fingerprinting
The Y chromosome:
1)  The Y chromosome changed in
such a way as to inhibit the
areas around the sex
determining genes from
recombining at all with the X
chromosome.
2)  95% of the human Y
chromosome is unable to
recombine and is passed on to
the next generation intact.
3)  Lack of recombination, makes
the Y chromosome a superb tool
for investigating recent human
evolution from a male
perspective.
Chemistry & Biochemistry – F2014
Chemistry & Biochemistry – F2014
7 9/30/14 Manipulating DNA sequences in organisms - Transgenics
Chemistry & Biochemistry – F2014
8