Nucleic Acid Chemistry

Where the info is…interpreting the

blueprint
 Friedrich Miescher in 1869

• Isolated what he called

nuclein from the nuclei of
pus cells
• Nuclein was shown to
have acidic properties,
hence it became called
nucleic acid
 Deoxyribonucleic acid,
DNA
Nucleic acid
Ribonucleic acid, RNA
 DNA as genetic material: The
circumstantial evidence
1. Present in all cells and virtually restricted
to the nucleus
2. The amount of DNA in somatic cells
(body cells) of any given species is
constant (like the number of
chromosomes)
3. The mutagenic effect of UV light peaks at
253.7nm. The peak for the absorption of
UV light by DNA
 The distribution of nucleic acids
in the eukaryotic cell
• DNA is found in the nucleus
with small amounts in mitochondria and
chloroplasts
• RNA is found throughout the cell

© 2016 Paul Billiet ODWS

 1. The components of DNA and
RNA
•DNA and RNA are polymers of nucleotide
units.
• DNA (RNA) consists of 4 kinds of
ribonucleotide units linked together through
covalent bonds.
• Each nucleotide unit is composed of
a nitrogenous base
a pentose sugar
a phosphate group
 NUCLEOTIDE STRUCTURE

PHOSPHATE SUGAR BASE

Ribose or PURINES PYRIMIDINES
Deoxyribose
Adenine (A) Cytocine (C)
Guanine(G) Thymine (T)
Uracil (U)

NUCLEOTIDE
© 2016 Paul Billiet ODWS
1.2 Ribose (in RNA) and deoxyribose (in DNA)
•

• Ribose and deoxyribose predominantly

exist in the cyclic form.
 1.1 Bases
• Purines :
– Adenine (A)
– Guanine (G)
• Pyrimidines :
– Cytosine (C)
– Uracil (U)
– Thymine (T)

DNA: A,G,C,T
RNA: A,G,C,U Thymine (T) is a 5-methyluracil (U)
1.3 Nucleosides =ribose/deoxyribose +
bases
•The bases are covalently attached to the 1’ position of a pentose
sugar ring, to form a nucleoside

Glycosidic bond

R
Ribose or 2’-deoxyribose
Adenosine, guanosine, cytidine, thymidine, uridine
1.4 Nucleotides = nucleoside +
phosphate
•A nucleotide is a nucleoside with one or more phosphate groups bound
covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the
case of 5’-position, up to three phosphates may be attached.

Phosphate ester bonds

Deoxynucleotides Ribonucleotides
(containing deoxyribose) (containing ribose)
 P
G

ADDING IN THE BASES P

C

• The bases are attached to

P
the 1st Carbon C
• Their order is important
P
It determines the genetic A
information of the molecule
P
T

P
© 2016 Paul Billiet ODWS
T
BASES NUCLEOSIDES NUCLEOTIDES
Adenine (A) Adenosine Adenosine 5’-triphosphate (ATP)
Deoxyadenosine Deoxyadenosine 5’-triphosphate
(dATP)
Guanine (G) Guanosine Guanosine 5’-triphosphate (GTP)
Deoxyguanosine Deoxy-guanosine 5’-triphosphate
(dGTP)
Cytosine (C) Cytidine Cytidine 5’-triphosphate (CTP)
Deoxycytidine Deoxy-cytidine 5’-triphosphate
(dCTP)
Uracil (U) Uridine Uridine 5’-triphosphate (UTP)

Thymine (T) Thymidine/ Thymidine/deoxythymidie

Deoxythymidie 5’-triphosphate (dTTP)
 phosphate
nucleic acid nucleotides pentose
nucleosides
bases

NH2

N
O
HO P O CH2 N O
O
OH

OH OH
 Composition of DNA and RNA

Nucleic
base ribose
acid

DNA A、G、C、T deoxyribose

RNA A、G、C、U ribose

 2. Structure and function of DNA

2.1 Primary structure

 Definition: the base sequence (or the
nucleotide sequence) in
polydeoxynucleotide chain.
 The smallest DNA in nature is virus DNA.
The length of φX174 virus DNA is 5,386
bases (a single chain).
 The DNA length of human genome is
3,000,000,000 pair bases.
5’end

Phosphodiester
bond

3’ end: free hydroxyl

(-OH) group

• 3’,5’ phosphodiester bond link nucleotides

together to form polynucleotide chains
The structure of a DNA chain can
be concisely represented

• An even more abbreviated notation for

this chain is
– pApCpGpTpA
– pACGTA
• The base chain is written in the 5’ →3’
direction
2.2 Secondary structure

The secondary structure is defined as the

relative spatial position of all the atoms of
nucleotide residues.
Secondary structure
— DNA double helix structure

Francis H.C. Crick

•Watson and Crick , 1953
•The genetic material of
all organisms except for
some viruses.
•The foundation of the
molecular biology.

James D. Watson
 The discovery of DNA double
helix
• Chargaff's Rule
(A=T, G=C in DNA)

• Franklin, Wilkins:
X-ray Diffraction
Refined Structure
 DNA IS MADE OF TWO STRANDS OF
POLYNUCLEOTIDE
• The sister strands of the DNA molecule run in
opposite directions (antiparallel)
• They are joined by the bases
• Each base is paired with a specific partner:
A is always paired with T
G is always paired with C
Purine with Pyrimidine
• Thus the sister strands are complementary but not
identical
• The bases are joined by hydrogen bonds,
individually weak but collectively strong.
© 2016 Paul Billiet ODWS
 DNA double helix Essential for replicating DNA and
•Two separate strands transcribing RNA
•Antiparellel (5’3’
direction) 3’
•Base pairing: hydrogen 5’
bonding that holds two
strands together
•Complementary (sequence)

• Sugar-phosphate backbones
(negatively charged): outside
• Base pairs (stack one above the
other): inside

3’
5’
 4
3 2 1
7 6
8 5 1
9
4
3 2

A:T G:C

Base
pairing
 B form of DNA double helix
• Right-handed helix;
•The diameter of the double
helix ： 2 nm
• The distance between two
base pairs: 0.34 nm;
• Each turn of the helix
involves 10 bases pairs, 3.4
nm.

 Stable configuration can be

maintained by hydrogen bond
and base stacking force
(hydrophobic interaction).
 Groove binding

• Small molecules like drugs bind in the minor

groove, whereas particular protein motifs can
interact with the major grooves.
• Watson, Crick, and Wilkins
shared the Nobel Prize in
medicine or physiology in 1962
for this brilliant
accomplishment.

• The discovery of the DNA

double helix revolutionized
biology: it led the way to an
understanding of gene function
in molecular terms (their work is
recognized to mark the
beginning of molecular biology).
Conformational variation in
double-helical structure
• B-DNA
• A-DNA
• Z-DNA
• B-form: the duplex structure proposed by Watson and Crick is referred as
the B-form DNA.
•It is the standard structure for DNA molecules.
•A-form: at low humidity the DNA molecule will take the A-form:
•The A-form helix is wider and shorter, with a shorter more
compact helical structure, than the B-form helix.
• Z-form: the Z-form DNA is adopted by short oligonucleotides.
•It is a left-handed double helix in which backbone phosphates
zigzag.
 2.3 Tertiary structure :
• Supercoils: double-stranded circular DNA
form supercoils if the strands are
underwound (negatively supercoiled) or
overwound (positively supercoiled).

Increasing degree of supercoiling

Relaxed supercoiled
• If the strands
are overwound,
form positively
supercoiled;
• If the strands
are underwound,
form negatively
supercoiled.
• The DNA in a prokaryotic cell is a
supercoil.

• Supercoiling makes the DNA molecule more

compact thus important for its packaging
in cells.
2.4 Eukaryotic DNA

• DNA in eukaryotic cells is highly

packed.
• DNA appears in a highly ordered form
called chromosomes during metaphase,
whereas shows a relatively loose form
of chromatin in other phases.
• The basic unit of chromatin is
nucleosome.
• Nucleosomes are composed of DNA
and histone proteins.
 The importance of packing of DNA
into chromosomes
 Chromosome is a compact form of the DNA
that readily fits inside the cell
 To protect DNA from damage
 DNA in a chromosome can be transmitted
efficiently to both daughter cells during
cell division
 Chromosome confers an overall organization
to each molecule of DNA, which facilitates
gene expression as well as recombination.
 2.5 Functions of
DNA
• The carrier of genetic information.
• The template strand involved in replication and
transcription.

Gene: the minimum functional unit in DNA

Genome: the total genes in a living cell or

living beings.
3. Structures and functions of
RNA
• Conformational variability of RNA is important
for the much more diverse roles of RNA in the
cell, when compared to DNA.

• Types :
• mRNA: messenger RNA, the carrier of genetic information
from DNA to translate into protein
• tRNA: transfer RNA , to transport amino acid to
ribosomes to synthesize protein
• rRNA: ribosomal RNA, the components of ribosomes
• hnRNA: Heterogeneous nuclear RNA
• snRNA: small nuclear RNA
Classes of eukaryotic RNAs
 RNA structure
• RNA molecules are largely single-stranded
but there are double-stranded regions.
4. Physical and Chemical
Properties of Nucleic Acids
 General properties
• Acidity
– Amphiphilic molecules; normally acidic because of
phosphate.
• Viscosity
– Solid DNA: white fiber; RNA: white powder.
Insoluble in organic solvents, can be precipitate by
ethanol.
• Optical absorption
– UV absorption due to aromatic groups.
• Thermal stability
– Disassociation of dsDNA (double-stranded DNA)
into two ssDNAs (single-stranded DNA).
 4.1 UV Absorption
• Specific absorption at 260nm.
• This can be used to identify nucleic
acid.

The UV absorption spectra of the common ribonucleotides

 4.2 Denaturation

• Concept:
• The course of hydrogen bonds
broken, 3-D structure was destroyed, the
double helix changed into single strand
irregular coil.
• Results:
(1) the value of 260nm absorption is
increased;
(2) biological functions are lost.
 • Heat denaturation and Tm

• When DNA were

heated to certain
temperature, the
absorption value at 260nm
would increased
sharply ， which indicates
that the double strand
helix DNA was separated
into single strand.

•Tm (melting temperature of DNA):

• The temperature of UV absorption increase to an half of maximum
value in DNA denaturation.
• Factors affect Tm:
G-C content:
•There are three hydrogen bonds between G-C pair. The more G-C
content, the higher Tm value.

Less G+C
Tm of
Higher G+C two DNA
molecules with
different G+C
content

Temperature
 4.3 Renaturation of DNA
• When slowly cooling down (Annealing)
the denatured DNA solution, the single
strand DNA can reform a double strands
helix to recover its biological functions.
 Molecule hybridization
• During the course of
lowing down denaturing
temperature, between
different resource DNAs
or single stand DNA and
RNA with
complementary bases
will repair into a double
strands to form a hybrid
DNA or DNA-RNA . This
course is called molecule
hybridization.
 Points
• The components of DNA and RNA
– Nucleotide: base (A,G,C,T,U), pentose sugar (Ribose
and deoxyribose), phosphate group
• Structure and function of DNA
– Primary structure: 3’,5’ phosphodiester bond
– Secondary structure: DNA double helix
– Tertiary structure: supercoil
– Eukaryotic chromosomes: nucleosome
• Structures and functions of RNA
– mRNA, tRNA, rRNA
• Properties of nucleic acid
– UV absorption, denaturation and renaturation, molecule
hybridization
 Central Dogma

Replication

DNA ---------------- RNA-------------- protein

transcription translation
 Central Dogma
• Replication
– DNA making a copy of itself
• Making a replica
• Transcription
– DNA being made into RNA
• Still in nucleotide language
• Translation
– RNA being made into protein
• Change to amino acid language
 Replication
• Remember that DNA is self
complementary
• Replication is semiconservative
– One strand goes to next generation
– Other is new
• Each strand is a template for the other
– If one strand is 5’ AGCT 3’
– Other is: 3’ TCGA 5’
Replication is Semiconservative
 Replication
• Roles of enzymes
– Topoisomerases
– Helicase
– DNA polymerases
– ligase
• DNA binding proteins
– DNA synthesis
• Leading strand
• Lagging strand
Replication
 Replication
• Helix opens
– Helicase
• Causes supercoiling upstream
– Topoisomerases (gyrase)
• DNA Binding Proteins
– Prevent reannealing
Replication
 Replication
• Leading strand
– 3’ end of template
– As opens up, DNA polymerase binds
– Makes new DNA 5’ - 3’
• Same direction as opening of helix
• Made continuously
 Replication
• Lagging strand
– 5’ end of template
• Can’t be made continuously as direction is wrong
– RNA primer
– New DNA made 5’  3’
• Opposite direction of replication
• Discontinuous
– Okazaki fragments
• Ligase closes gaps
 Transcription
• DNA template made into RNA copy
– Uracil instead of Thymine
• One DNA strand is template
– Sense strand
• Other is just for replication
– Antisense (not to be confused with
nonsense!)
• In nucleus
– nucleoli
Transcription
 Translation
• RNA -- Protein
– Change from nucleotide language to amino
acid language
• On ribosomes
• Vectorial nature preserved
– 5’ end of mRNA becomes amino terminus of
protein
– Translation depends on genetic code
 Genetic Code
• Nucleotides read in triplet “codons”
– 5’ - 3’
• Each codon translates to an amino acid
• 64 possible codons
– 3 positions and 4 possiblities (AGCU) makes
43 or 64 possibilities
– Degeneracy or redundancy of code
• Only 20 amino acids
• Implications for mutations
Genetic Code
 Genetic Code
• Not everything translated
• AUG is start codon
– Find the start codon
• Also are stop codons
• To determine aa sequence
– Find start codon
– Read in threes
– Continue to stop codon
 Translation
• Initiation
– Ribosomal subunits assemble on mRNA
– rRNA aids in binding of mRNA
• Elongation
– tRNAs with appropriate anticodon loops bind to complex
– have aa attached (done by other enzymes)
– Amino acids transfer form tRNA 2 to tRNA 1
– Process repeats
• Termination
– tRNA with stop codon binds into ribosome
– No aa attached to tRNA
– Complex falls apart
Translation
GENETIC ENGINEERING
 Genetic Engineering
• Any process by which genetic material is
changed in such a way as to make
possible of the production of new
substances or new functions.
Chemical Structure of Genes
Each DNA has a pattern

CODONS (amino acids)

Arranged into
particular sequence

protein
 Gene Splicing
• Is the process in which fragments of DNA
from one or more different microorganism are
combined to form rDNA (recombinant DNA)
and are made to function within the cell of a
host organism.

• 2 highly significant techniques:

– Gene transfer
• transferring the gene from one source to another
subject.
– Gene therapy
• Correcting defective gene that are responsible for
disease development.
Gene Splicing
 Gene Splicing
• Plasmid - A circular form of DNA often used as a vector in genetic
engineering.
• Vector – an organism/ chemical that is used to transport a gene to
the host cell.
• Host cell – the cell into where the new gene is transplanted

• Enzymes used:
– Endonucleases – enzymes that cut DNA molecule at some given
location
– Exonucleases – enzyme that removes one nitrogen base unit at a time
– Ligases – enzyme that join two DNA segments together
 Gene Transfer
Although the concept of gene transfer is relatively
simple, its execution presents considerable
technical obstacles.

 American biochemist Paul Berg (1926-), often referred

to as the “father of genetic engineering”.
 He developed a method for joining the DNA from two different
organisms, a monkey virus known as SV40 and a virus
called lambda phage.

• the American biochemists Stanley Cohen (1922-) at Stanford

University, and Herbert Boyer (1936-) at the University of California
and San Francisco, discovered an enzyme that greatly increased
the efficiency of the Berg procedure.
 Gene Transfer
• INSULIN
– Produced by “Genetech”, first genetic
engineering company, founded by Robert
Swanson and Herbert Boyer.
• Obtains a copy of insulin gene (can be from natural
source or manufactured)
• Inserting the insulin gene into the vector (using the
gene splicing process)
• The hybrid plasmid can now be inserted to the host
cell. ( this is the manufactured insulin that is injected
to diabetic patients)
 Other rDNA products
• Human growth hormone
– For children whose growth is insufficient bc of genetic
problems
• Interleukin-2
– For treatment of cancer
• Factor VIII
– Needed by hemophiliacs for blood clotting
• Erythropoietin
– For treatment of anemia
• Tumor necrosis factor
– For treatment of tumors
• Tissue plasminogen activator
– Use to dissolve blood clots
 Gene Therapy
• 4 approaches
– A normal gene inserted to compensate for the
defective gene.
– Abnormal gene replaced with a normal one
– Abnormal gene repaired through selective
reverse mutation
– Change the regulation of gene pairs.
Gene Therapy
 Gene Therapy
– A vector delivers the therapeutic gene into a
patient’s target cell
– The target cells become infected with the viral
vector
– The vector’s genetic material is inserted into
the target cell
– Functional proteins are created from the
therapeutic gene causing the cell to return to
a normal state.
 Gene Therapy
• The first gene therapy was performed on
September 14th 1990
– Ashanti DeSilva was treated for SCID
– Doctors removed her white blood cells,
inserted the missing gene into the WBC and
then put them back into her blood stream
– It strengthened her immune system, but it
only worked for a few months
 Advantages
– Genetic Engineering could increase genetic
diversity, and produce more variant alleles which
could also be crossed over and implanted into other
species
– Another of genetic engineering is that diseases
could be prevented by detecting people that are
genetically prone to certain hereditary diseases, and
preparing for the inevitable. As well as preventing
disease, with genetic engineering infectious
diseases can be treated by implanting genes that
code for antiviral proteins specific to each antigen
 Advantages
– Animals and plants can be 'tailor made' to
show desirable characteristics. Genes could
also be manipulated in trees for example, to
absorb more CO2 and reduce the threat of
global warming.
 Disadvantages

– Nature is an extremely complex inter-related

chain consisting of many species linked in the
food chain. Some scientists believe that
introducing genetically modified genes may have
an irreversible effect with consequences yet
unknown.
– Genetic engineering borderlines on many moral
issues, particularly involving religion, which
questions whether man has the right to
manipulate the laws and course of nature.