Nucleic acid LEARNING OBJECTIVES:

• Nucleotide Structure
• Locate the structural components of DNA, namely, the
nitrogenous bases, the sugar, and the phosphate group.
Know the various conventions used to represent these
components and the structure of DNA
• Differentiate purines, pyrimidines, ribonucleosides,
deoxyribonucleosides, ribonucleotides, and
deoxyribonucleotides.
• Recognize the deoxyadenosine, deoxycytidine,
deoxyguanosine, and deoxythymidine constituents of DNA,
and describe the phosphodiester bond that joins them
together to form DNA.
• Compare the phosphodiester backbones of RNA and DNA.
Contrast the composition and structures of RNA and DNA.
Which of the preceding structures (a) contains ribose? (b) contains
deoxyribose? (c) contains a purine? (d) contains a pyrimidine? (e) contains
guanine? (f) contains a phosphate monoester? (g) contains a
phosphodiester? (h) is a nucleoside? (i) is a nucleotide? (j) would be found in
RNA? (k) would be found in DNA?
• Nucleotides are synthesized from many simple precursors
and the pentose, phosphoribosyl pyrophosphate.
• Nucleotides function as energy carriers and regulatory
molecules and as precursors to coenzymes and nucleic
acids.
 Nucleic acid
• Group of complex compounds consisting of linear
chains of monomeric nucleotides.
• Each monomeric unit is composed of phosphoric
acid, sugar and nitrogenous base (a purine or
pyrimidine),
• Involved in the preservation, replication, and
expression of hereditary information in every living
cell.
 Nucleic acid

❑ Nucleic acids are polynucleotides—that is, long

chainlike molecules composed of a series of
nearly identical building blocks called
nucleotides.
❑ Each nucleotide consists of a nitrogen-
containing aromatic base attached to a pentose
(five-carbon) sugar, which is in turn attached to
a phosphate group.
 Nucleic acid

Basically, nucleic acids can be subdivided into

two types:
❑ Deoxyribonucleic acid (DNA) and
❑ Ribonucleic acid (RNA).
 Nucleic acid

Both DNA and RNA have been shown to consist

of three groups of molecules:

❑ Pentose (5-carbon-atom) sugars;

❑ Nitrogen containing organic bases; and
❑ Inorganic phosphate.
 Bases

Each nucleic acid contains four of five possible

nitrogen-containing bases:
1. Adenine (A),
2. Guanine (G),
3. Cytosine (C),
4. Thymine (T), and
5. Uracil (U).
 Bases

❑ A and G are categorized as purines, and

❑ C, T, and U are collectively called pyrimidines.
❑ All nucleic acids contain the bases A, C, and G;
❑ T is found only in DNA,
❑ while U is found only in RNA.
Bases
 There are five common bases, and four are generally
represented in either DNA or RNA. Those bases and their
corresponding nucleosides are described in the following table:

Abbr. Base Nucleoside Nucleic Acid

deoxyadenosine DNA
A Adenine
adenosine RNA
deoxyguanosine DNA
G Guanine
guanosine RNA
deoxycytidine DNA
C Cytosine
cytidine RNA
deoxythymidine
T Thymine DNA
(thymidine)
U Uracil uridine RNA
 Sugar

❑ There are only two types of sugar present in

nucleic acids.
❑ Ribose which is present solely in RNA (hence
its name ribonucleic acid ) and
❑ Deoxyribose which is present solely in DNA
(again, the sugar gives rise to the name
deoxyribonucleic acid).
 Sugar
The chemical structures for these compounds
are shown here:
 Sugar

❑ The pentose sugar in DNA is 2′-deoxyribose.

❑ The sugar in RNA is 2′-ribose.
❑ 2′-deoxyribose differs from 2′-ribose by the
absence of a hydroxyl group (−OH) on the 2′
carbon of the sugar ring.
 Sugar
❑ The prefix ‘deoxy’ means ‘without oxygen’,
❑ From the structures, the only difference
between them is the absence of an oxygen in the
deoxyribose sugar.
❑ Both sugars contain 5 carbon atoms (pentose
sugars) and for convenience we number these as
shown in the figure.
 Sugar

❑ The ‘dash’ or ‘prime’ (') on, for example, the 5

indicates the carbon in the ribose ring.
❑ The carbon atoms are numbered 1', 2', 3', 4',
and 5' (prime) to distinguish from the
numbering of the atoms of the purine and
pyrmidine rings.
 Sugar

❑ The hydroxyl groups on the 5'- and 3'- carbons

link to the phosphate groups to form the DNA
backbone.
❑ Deoxyribose lacks an hydroxyl group at the 2'-
position when compared to ribose, the sugar
component of RNA.
 Inorganic phosphate

There are phosphate residues in nucleic acids and

they are of the type derived from phosphoric acid,
the structure of which is shown below.
Building nucleic acids from their building blocks

When any one of the bases is joined to either

one of the two sugar molecules, a new
compound is formed known as a nucleoside.
Building nucleic acids from their building blocks

❑ If the sugar residue is ribose then we have a

ribonucleoside, whereas if it is deoxyribose
then we have a deoxyribonucleoside.
❑ The bond linking these structures is known
as a glycoside bond.
Building nucleic acids from their building blocks
Building nucleic acids from their building blocks
 There are five common bases, and four are generally
represented in either DNA or RNA. Those bases and their
corresponding nucleosides are described in the following table:

Abbr. Base Nucleoside Nucleic Acid

deoxyadenosine DNA
A Adenine
adenosine RNA
deoxyguanosine DNA
G Guanine
guanosine RNA
deoxycytidine DNA
C Cytosine
cytidine RNA
deoxythymidine
T Thymine DNA
(thymidine)
U Uracil uridine RNA
Building nucleic acids from their building blocks

❑ Addition of a phosphate group to the sugar

residue of a nucleoside molecule produces a
different molecule called a nucleotide.
❑ Nucleotides can be regarded as the building
blocks for the larger nucleic acid molecules,
DNA and RNA.
Building nucleic acids from their building blocks

Examples of various nucleotides are shown below.

Building nucleic acids from their building blocks

❑ One or two additional phosphates can be added

to the first phosphate group of a nucleoside
molecule (a nucleoside monophosphate, NMP)
by means of a pyrophosphate linkage.
❑ The molecules formed in this way are called
nucleoside diphosphates (NDPs) and nucleoside
triphosphates (NTPs).
Building nucleic acids from their building blocks
Building nucleic acids from their building blocks
Building nucleic acids from their building blocks

The most important base involved in these

compounds is adenine, forming the adenosine
mono-, di- and triphosphate molecules (AMP, ADP
and ATP), which fulfill vital roles in many cellular
processes.
Building nucleic acids from their building blocks

❑ Nucleic acids are formed by the combination of

nucleotide molecules through sugar–phosphate
bonds known as phosphodiester linkages.
❑ Because a nucleic acid is a polymer of many
nucleotide molecules, DNA and RNA molecules
are called polynucleotides.
The structure of a polynucleotide is shown diagrammatically below.
 DNA -Deoxyribonucleic acid

The nucleic acid that stores and transmits

the genetic information from one generation
of an organism to the next.
 Chemical Nature of DNA
❑ The polymeric structure of DNA may be
described in terms of monomeric units of
increasing complexity.
❑ In the top shaded box of the following
illustration, the three relatively simple
components are shown.
❑ Below that on the left , formulas for
phosphoric acid and a nucleoside are
drawn.
Chemical Nature of DNA
 Chemical Nature of DNA
❑ The combination of a base and sugar is called a
nucleoside.
❑ Nucleotides also exist in activated forms
containing two or three phosphates, called
nucleotide diphosphates or triphosphates.
❑ If the sugar in a nucleotide is deoxyribose, the
nucleotide is called a deoxynucleotide; if the
sugar is ribose, the term ribonucleotide is used.
❑ The structure on the left - deoxyguanosine -
depicts the base, sugar and phosphate moieties.
Chemical Nature of DNA
 Nucleosides of DNA
❑ A nucleoside is one of the four DNA bases
covalently attached to the C1' position of a
sugar.
❑ The sugar in deoxynucleosides is 2'-deoxyribose.
❑ The sugar in ribonucleosides is ribose.
❑ Nucleosides differ from nucleotides in that they
lack phosphate groups.
❑ The four different nucleosides of DNA are
deoxyadenosine (dA), deoxyguanosine (dG),
deoxycytosine (dC), and (deoxy)thymidine (dT,
or T).
 Chemical Nature of DNA

❑ Condensation polymerization of these leads to

the DNA formulation outlined below.
❑ Finally, a 5'- monophosphate ester, called a
nucleotide may be drawn as a single monomer
unit, shown in the shaded box to the right.
❑ Names for these DNA components are given in
the following table.
Chemical Nature of DNA
Names of DNA Base Derivatives
Base Nucleoside 5'-Nucleotide

2'-Deoxyadenosine-
Adenine 2'-Deoxyadenosine
5'-monophosphate
2'-Deoxycytidine-
Cytosine 2'-Deoxycytidine
5'-monophosphate
2'-Deoxyguanosine-
Guanine 2'-Deoxyguanosine
5'-monophosphate
2'-Deoxythymidine-
Thymine 2'-Deoxythymidine
5'-monophosphate
Chemical Nature of DNA
 Chemical Nature of DNA

❑ Isomeric 3'-monophospate nucleotides are also

known, and both isomers are found in cells.
❑ They may be obtained by selective hydrolysis of
DNA through the action of nuclease enzymes.
❑ Anhydride-like di- and tri-phosphate
nucleotides have been identified as important
energy carriers in biochemical reactions, the
most common being ATP (adenosine 5'-
triphosphate).
A complete structural representation of a segment of the DNA
polymer formed from 5'-nucleotides may be viewed on the
below diagram.
 Several important characteristics of this formula:

❑ First, the remaining P-OH function is quite acidic and is

completely ionized in biological systems.
❑ Second, the polymer chain is structurally directed. One
end (5') is different from the other (3').
❑ Third, although this appears to be a relatively simple
polymer, the possible permutations of the four
nucleosides in the chain become very large as the chain
lengthens.
❑ Fourth, the DNA polymer is much larger than originally
believed. Molecular weights for the DNA from
multicellular organisms are commonly 109 or greater.
Several important characteristics of this formula:

❑ Information is stored or encoded in the DNA

polymer by the pattern in which the four
nucleotides are arranged.
❑ To access this information the pattern must be
"read" in a linear fashion, just as a bar code is
read at a supermarket checkout.
❑ Because living organisms are extremely
complex, a correspondingly large amount of
information related to this complexity must be
stored in the DNA.
Several important characteristics of this formula:

❑ Consequently, the DNA itself must be very

large, as noted above.
❑ Even the single DNA molecule from an E. coli
bacterium is found to have roughly a million
nucleotide units in a polymer strand, and would
reach a millimeter in length if stretched out.
❑ The nuclei of multicellular organisms
incorporate chromosomes, which are composed
of DNA combined with nuclear proteins called
histones.
Several important characteristics of this formula:

❑ The fruit fly has 8 chromosomes, humans have 46

and dogs 78 (note that the amount of DNA in a
cell's nucleus does not correlate with the number
of chromosomes).
❑ The DNA from the smallest human chromosome
is over ten times larger than E. coli DNA, and it
has been estimated that the total DNA in a human
cell would extend to 2 meters in length if
unraveled.
❑ Since the nucleus is only about 5μm in diameter,
the chromosomal DNA must be packed tightly to
fit in that small volume.
Several important characteristics of this formula:

❑ In addition to its role as a stable informational

library, chromosomal DNA must be structured or
organized in such a way that the chemical
machinery of the cell will have easy access to that
information, in order to make important molecules
such as polypeptides.
❑ Furthermore, accurate copies of the DNA code
must be created as cells divide, with the replicated
DNA molecules passed on to subsequent cell
generations, as well as to progeny of the organism.
❑ The nature of this DNA organization, or secondary
structure, will be discussed in a later section.
 Chemical Nature of DNA

❑ The phosphate group connects successive sugar

residues by bridging the 5′-hydroxyl group on
one sugar to the 3′-hydroxyl group of the next
sugar in the chain.
❑ These nucleoside linkages are called
phosphodiester bonds and are the same in RNA
and DNA.
Chemical Nature of DNA
 Chemical Nature of DNA
❑ In comparison, the structure on the right has an
extra hydroxyl group on the 2' carbon of ribose,
making it a ribonucleotide - riboguanosine or
just guanosine.
❑ In the right-hand figure, note also the 5' and 3'
carbons on ribose (or deoxyribose) -
understanding this concept and nomenclature is
critical to understanding polarity of nucleic
acids, as discussed below.
❑ The 5' carbon has an attached phosphate group,
while the 3' carbon has a hydroxyl group.
 Nucleic Acids
❑ DNA and RNA are synthesized in cells by DNA
polymerases and RNA polymerases.
❑ Short fragments of nucleic acids also are
commonly produced without enzymes by
oligonucleotide synthesizers.
❑ In all cases, the process involves forming
phosphodiester bonds between the 3' carbon of
one nucleotide and the 5' carbon of another
nucleotide.
❑ This leads to formation of the so-called "sugar-
phosphate backbone", from which the bases
project.
Nucleic Acids
 Nucleic Acids
❑ A key feature of all nucleic acids is that they have two
distinctive ends: the 5' (5-prime) and 3' (3-prime)
ends.
❑ This terminology refers to the 5' and 3' carbons on the
sugar.
❑ For both DNA (shown above) and RNA, the 5' end
bears a phosphate, and the 3' end a hydroxyl group.
❑ Another important concept in nucleic acid structure
is that DNA and RNA polymerases add nucleotides to
the 3' end of the previously incorporated base.
❑ Another way to put this is that nucleic acids are
synthesized in a 5' to 3' direction.
 Base Pairing and Double Stranded Nucleic Acids

❑ Most DNA exists in the famous form of a double

helix, in which two linear strands of DNA are
wound around one another.
❑ The major force promoting formation of this
helix is complementary base pairing:
❑ A's form hydrogen bonds with T's (or U's in
RNA), G's form hydrogen bonds with C's.
❑ If we mix two ATGC's together, the following
duplex will form:
Base Pairing and Double Stranded Nucleic Acids
Examine the figure above and note two very
important features:
1. The two strands of DNA are arranged
antiparallel to one another: viewed from left
to right the "top" strand is aligned 5' to 3',
while the "bottom" strand is aligned 3' to 5'.
This is always the case for duplex nucleic
acids.
2. G-C base pairs have 3 hydrogen bonds,
whereas A-T base pairs have 2 hydrogen
bonds: one consequence of this disparity is
that it takes more energy (e.g. a higher
temperature) to disrupt GC-rich DNA than
AT-rich DNA.
Examine the figure above and note two very
important features:

❑ The figures above fail to impart any

appreciation of the three-dimensional structure
of DNA.
❑ This deficiency can be rectified to some extent
by viewing and manipulating a 3-D model of
duplex DNA.
 Base-pairing rules

❑ The base-pairing rules are followed throughout

the steps of the central dogma.
❑ In transcription, nucleotides from DNA pair
with nucleotides with RNA.
❑ In this case, guanine (G) still pairs with cytosine
(C).
❑ However, because RNA does not contain
thymine (T), adenine (A) in DNA pairs with
uracil (U) in RNA.
 Base-pairing rules
❑ Later in the translation step of the central
dogma, RNA nucleotides pair with RNA
nucleotides.
❑ Again, guanine (G) pairs with cytosine (C) and
adenine (A) pairs with uracil (U).
❑ The base-pairing rules for each of these
situations are summarized in the following
table.
 Base-pairing rules
❑ Different types of nucleic acids are capable of
base-pairing with each other by following the
base-pairing rules.
❑ These rules are used throughout the process of
the central dogma to allow information to go
from DNA to RNA and then ultimately to the
amino acid sequence of proteins.
❑ In general, the rules are that G and C pair, and
A and T (or U) pair.
❑ Remember that thymine is only found in DNA
and uracil is found only in RNA.
 Base-pairing rules
❑ Although DNA is a double helix, it is often
depicted as a ladder.
❑ The base pairs make up the rungs of the ladder
and the alternating sugar and phosphate groups
make up the limbs of the ladder.
❑ Unlike a ladder, however, DNA strands are
arranged in an antiparallel manner.
❑ The 5' end of one strand is paired with the 3'
end of the other strand.
❑ And, of course, the two strands of a DNA double
helix are complementary (e.g., A pairs with T; G
with C).
 Base-pairing rules
There are several different ways to depict the
sequence to the right:

5' TCGTCA 3' 5’ TGACGA 3’

3' AGCAGT 5' 3’ ACTGCT 5’

TCGTCA TGACGA
AGCAGT ACTGCT

TCGTCA TGACGA
Some useful chemical properties of DNA

DNA is negatively charged

The phosphate backbone confers a net negative
charge on DNA, a property important for
electrophoretic analysis.
Some useful chemical properties of DNA

DNA can be denatured and renatured

By raising the temperature or using chemical
denaturants, it is possible to disrupt the hydrogen
bonds between bases on different strands and
“melt” the double helix into two polynucleotide
strands.
This process is called denaturation.
Some useful chemical properties of DNA

DNA can be denatured and renatured (contd.)

The process is reversible—if conditions are
favorable, the DNA strands can reanneal.
Renaturation of two strands occurs efficiently only
when the sequences of base pairs are
complementary, i.e., when A-T and G-C pairs can
form.
Association of complementary single-stranded
sequences is also called nucleic acid hybridization.
Some useful chemical properties of DNA

DNA is soluble in water

DNA is quite soluble in water and is generally kept
as a buffered solution.
Routinely, a buffer containing a small amount of
the buffer Tris (to control pH) and the chelating
agent EDTA (to trap cations which are cofactors
for enzymes that can attack DNA) is used to
solublize nucleic acids.
DNA is insoluble in ethanol.
Some useful chemical properties of DNA
DNA absorbs ultraviolet light
The purine and pyrimidine bases absorb light
strongly in the ultraviolet end of the spectrum,
with a maximum absorbance at 260 nm.
Over a range of concentrations, the amount of light
absorbed is proportional to the amount of DNA in
solution.
For double-stranded DNA, an A260 reading of 1.0
corresponds to a concentration of 0.05 mg/ml.
Thus, by measuring the absorbance of a DNA
solution of unknown concentration, one can
calculate the concentration of DNA in the solution.
Some useful chemical properties of DNA

DNA can be stained and quantified with ethidium

bromide
Ethidium bromide (EtBr) is a flat, ring-shaped
molecule that can insert between base pairs of the
double helix (see the figure on the next page).
This insertion, or intercalation, is useful for
visualization of DNA because EtBr fluoresces when
exposed to ultraviolet light.
Thus, DNA, normally invisible, can be seen and
photographed by staining with EtBr.
Some useful chemical properties of DNA
DNA can be stained and quantified with ethidium
bromide
Additionally, because the amount of fluorescence is
proportional to the total mass of DNA in a solution, the
quantity of DNA can be estimated by comparing the
fluorescent yield of the sample with that of a series of
standards.
The intercalation of ethidium bromide into a DNA
molecule is shown in the figure at the right.
Note that the ethidium bromide increases the spacing
of successive base pairs, distorts the regular sugar-
phosphate backbone, and decreases the pitch of the
helix.
DNA structure and function
 RNA-Ribonucleic acid
❑ A lower molecular weight, but much more
abundant nucleic acid, RNA, is distributed
throughout the cell, most commonly in small
numerous organelles called ribosomes.
❑ Three kinds of RNA are identified, the largest
subgroup (85 to 90%) being ribosomal RNA,
rRNA, the major component of ribosomes,
together with proteins.
❑ The size of rRNA molecules varies, but is
generally less than a thousandth the size of
DNA.
 RNA-Ribonucleic acid

❑ The other forms of RNA are messenger RNA,

mRNA, and transfer RNA, tRNA.
❑ Both have a more transient existence and are
smaller than rRNA.
❑ All these RNA's have similar constitutions, and
differ from DNA in two important respects.
❑ As shown in the following diagram, the sugar
component of RNA is ribose, and the pyrimidine
base uracil replaces the thymine base of DNA.
 RNA-Ribonucleic acid

❑ The RNA's play a vital role in the transfer of

information (transcription) from the DNA
library to the protein factories called ribosomes,
and in the interpretation of that information
(translation) for the synthesis of specific
polypeptides.
A complete structural representation of a segment of the RNA polymer
formed from 5'-nucleotides may be viewed on the below diagram
 Types of RNA

RNA has three major subtypes:

❑ Messenger RNA (mRNA),
❑ Transfer RNA (tRNA), and
❑ Ribosomal RNA (rRNA).

All three of those subtypes are involved in protein

synthesis.
 Types of RNA
Here is how a protein is made.
❑ First, messenger RNA (mRNA) goes into the
nucleus of the cell and copies the recipe for a
specific protein.
❑ The mRNA brings the recipe outside the nucleus
to the ribosome; the site of protein synthesis
inside the cell.
❑ Ribosomal RNA (rRNA) with the help of
transfer RNA (tRNA) synthesizes a single amino
acid.
 Types of RNA

❑ mRNA– Carries copies of instructions for the

assembly of amino acids into proteins from DNA
to the rest of the cell.
❑ rRNA– Makes up the major part of ribosomes.
❑ tRNA– Transfers amino acids to ribosome's
during protein synthesis.
 DNA vs RNA
Stands for:
Deoxyribo NucleicAcid
Ribo Nucleic Acid

DNA contains the sugar deoxyribose.

RNA contains the sugar ribose.
The only difference between ribose and
deoxyribose is that ribose has one more -OH group
than deoxyribose, which has -H attached to the
second (2') carbon in the ring.
 DNA vs RNA
If the prefix “de-“ means “without one” and “oxy-“
refers to oxygen then deoxyribose has the same
structure as ribose except that it has one less oxygen.
(literally, “without one oxygen”)
 DNA vs RNA

DNA is a double stranded molecule

RNA is a single stranded molecule

DNA is stable under alkaline conditions

RNA is not stable under alkaline conditions
 DNA vs RNA

DNA is responsible for storing and transferring

genetic information
RNA directly codes for amino acids and as acts as a
messenger between DNA and ribosomes to make
proteins.
 DNA vs RNA

DNA uses the bases adenine, thymine, cytosine, and

guanine;
RNA uses adenine, uracil, cytosine, and guanine.

Uracil differs from thymine in that it lacks a

methyl group on its ring.
 DNA vs RNA

Predominant Structure:
Double- stranded molecule with a long chain of
nucleotides
A single-stranded molecule in most of its biological
roles and has a shorter chain of nucleotides
 DNA vs RNA

Pairing of Bases:
A-T(Adenine-Thymine), G-C(Guanine-Cytosine)
A-U(Adenine-Uracil), G-C(Guanine-Cytosine)
 DNA vs RNA

Stability:
Deoxyribose sugar in DNA is less reactive because
of C-H bonds. Stable in alkaline conditions. DNA
has smaller grooves, which makes it harder for
enzymes to "attack" DNA.
Ribose sugar is more reactive because of C-OH
(hydroxyl) bonds. Not stable in alkaline conditions.
RNA has larger grooves, which makes it easier to
be attacked by enzymes.
 DNA vs RNA

Propagation:
DNA is self-replicating.
RNA is synthesized from DNA when needed.
 DNA vs RNA

Bases & Sugars:

Deoxyribose sugar; phosphate backbone; Four
bases: adenine, guanine, cytosine and thymine
Ribose sugar; phosphate backbone; Four bases:
adenine, guanine, cytosine, and uracil
 DNA vs RNA

Job/Role:
Medium of long-term storage and transmission of
genetic information
Transfer the genetic code needed for the creation of
proteins from the nucleus to the ribosome.
 DNA vs RNA

Unique Features:
The helix geometry of DNA is of B-Form. DNA is
completely protected by the body, i.e., the body
destroys enzymes that cleave DNA. DNA can be
damaged by exposure to Ultra-violet rays
The helix geometry of RNA is of A-Form. RNA
strands are continually made, broken down and
reused. RNA is more resistant to damage by Ultra-
violet rays.
 DNA vs RNA
Definition:
A nucleic acid that contains the genetic instructions
used in the development and functioning of all
modern living organisms (scientists believe that
RNA may have been the main genetic material in
primitive life forms).
A single-stranded chain of alternating phosphate
and ribose units with the bases Adenine, Guanine,
Cytosine, and Uracil bonded to the ribose. RNA
molecules are involved in protein synthesis and
sometimes in the transmission of genetic
information.
 DNA vs RNA
Ultraviolet Damage
DNA is susceptible to UV damage.
Compared with DNA, RNA is relatively resistant to
UV damage.

DNA is not found in the cytoplasm

RNA is found in the cytoplasm
 DNA vs RNA
Reactivity
The C-H bonds in DNA make it fairly stable, plus
the body destroys enzymes that would attack DNA.
The small grooves in the helix also serve as
protection, providing minimal space for enzymes to
attach.
The O-H bond in the ribose of RNA makes the
molecule more reactive, compared with DNA. RNA
is not stable under alkaline conditions, plus the
large grooves in the molecule make it susceptible to
enzyme attack. RNA is constantly produced, used,
degraded, and recycled.
 Role of Nucleic Acids
❑ The main function of nucleic acids is to
store and transmit genetic information
and use that information to direct the
synthesis of new protein.
❑ DNA (deoxyribonucleic acid) is the
permanent storage place for genetic
information in the nucleus of a cell.
❑ DNA controls the synthesis of RNA
(ribonucleic acid).
 Role of Nucleic Acids
❑ RNA transmits genetic information from
DNA to the protein synthesizers in the
cell.
❑ RNA is also responsible for directing the
production of the new protein by
transmitting the genetic information to
the protein building structures.
❑ The nucleotide ATP (adenosine
triphosphate), which is closely related to
DNA and RNA, is the short-term energy
storage for all life processes.
 Role of Nucleic Acids
❑ The function of the sequence of bases (adenine,
cytosine, guanine, and thymine) in the backbone
of DNA determine what proteins are being
synthesized and in what order (note that in
RNA, thymine is replaced by uracil).
❑ The function of the double helix formation of
DNA molecules is to ensure that no disorders
occur if genetic information is lost or damaged.
❑ Examples of disorders related to damaged or
lost genetic information are Down’s Syndrome
and Sickle Cell Anemia.
 Gene

❑ Certain sections of nitrogenous bases along the

strand of DNA form a gene.
❑ A gene is a unit that contains the genetic
information or codes for a particular product
and transmits hereditary information to the
next generation.
 The Double Helix
❑ After many trials and modifications, Watson
and Crick conceived an ingenious double helix
model for the secondary structure of DNA.
❑ The two strands are identical.
❑ Though the two strands are identical, they run
in opposite directions (anti-parallel to each
other) as determined by the orientation of the 5′
to 3′ phosphodiester bond.
❑ This is shown in part a of the following
diagram.
 The Double Helix
❑ The sugar-phosphate chains run along the
outside of the helix, and
❑ The bases lie on the inside,
❑ Where they are linked to complementary bases
on the other strand through hydrogen bonds.
❑ Complementary primary nucleotide structures
for each strand allowed intra-strand hydrogen
bonding between each pair of bases.
❑ These complementary strands are colored red
and green in the diagram.
 The Double Helix
❑ Coiling these coupled strands then leads to a
double helix structure, shown as cross-linked
ribbons in part b of the diagram.
❑ The double helix is further stabilized by
hydrophobic attractions and pi-stacking of the
bases.
❑ A space-filling molecular model of a short
segment is displayed in part c on the right.
❑ The helix shown here has ten base pairs per one
complete turn, and rises 3.4 Å in each turn.
 The Double Helix
❑ The double helical structure of normal DNA
takes a right-handed form.
❑ This right-handed helix is the favored
conformation in aqueous systems, and has been
termed the B-helix.
❑ As the DNA strands wind around each other,
they leave gaps between each set of phosphate
backbones.
❑ Two alternating grooves result, a wide and deep
major groove (ca. 22Å wide), and a shallow and
narrow minor groove (ca. 12Å wide).
 The Double Helix
❑ Many proteins interact in the space of the major
groove, where they make sequence-specific
contacts with the bases.
❑ In addition, a few proteins are known to make
contacts via the minor groove.
❑ Other molecules, including polypeptides, may
insert into these grooves, and in so doing
perturb the chemistry of DNA.
❑ Other helical structures of DNA have also been
observed, and are designated by letters (e.g. A
and Z).
The secondary structure of DNA

Francis Crick and James Watson, at Cambridge

University, considered hydrogen bonded base
pairing interactions, and arrived at a double
stranded helical model that satisfied most of the
known facts, and has been confirmed by
subsequent findings.
 Base Pairing

➢ Careful examination of the purine and

pyrimidine base components of the nucleotides
reveals that three of them could exist as hydroxy
pyrimidine or purine tautomers, having an
aromatic heterocyclic ring.
➢ Despite the added stabilization of an aromatic
ring, these compounds prefer to adopt amide-
like structures.
 Base Pairing
These options are shown in the following diagram,
with the more stable tautomer drawn in blue.
 Base Pairing
➢ A simple model for this tautomerism is provided
by 2-hydroxypyridine.
➢ As shown on the left below, a compound having
this structure might be expected to have phenol-
like characteristics, such as an acidic hydroxyl
group.
 Base Pairing
➢ This tautomerism reverses the hydrogen
bonding behavior of the nitrogen and oxygen
functions (the N-H group of the pyridone
becomes a hydrogen bond donor and the
carbonyl oxygen an acceptor).
 Base Pairing
➢ The boiling point of the actual substance is 100º
C greater than phenol and its acidity is 100
times less than expected (pKa = 11.7).
➢ These differences agree with the 2-pyridone
tautomer, the stable form of the zwitterionic
internal salt.
➢ Further evidence supporting this assignment
will be displayed on the following diagram.
 Base Pairing

➢ The additional evidence for the pyridone

tautomer, that appears on the diagram, consists
of infrared and carbon nmr absorptions
associated with and characteristic of the amide
group.
➢ The data for 2-pyridone is given on the left.
Similar data for the N-methyl derivative, which
cannot tautomerize to a pyridine derivative, is
presented on the right.
 Base Pairing
➢ Once they had identified the favored base
tautomers in the nucleosides, Watson and Crick
were able to propose a complementary pairing,
via hydrogen bonding, of guanosine (G) with
cytidine (C) and adenosine (A) with thymidine
(T).
➢ This pairing, which is shown in the following
diagram, explained Chargaff's findings
beautifully, and led them to suggest a double
helix structure for DNA.
Base Pairing
 Base Pairing

➢ Before viewing this double helix structure itself,

it is instructive to examine the base pairing
interactions in greater detail.
➢ The G#C association involves three hydrogen
bonds (colored pink), and is therefore stronger
than the two-hydrogen bond association of A#T.
➢ These base pairings might appear to be
arbitrary, but other possibilities suffer
destabilizing steric or electronic interactions.
 Base Pairing

➢In the following diagram two such alternative

couplings will be shown.
➢The C#T pairing on the left suffers from
carbonyl dipole repulsion, as well as steric
crowding of the oxygens.
➢The G#A pairing on the right is also destabilized
by steric crowding (circled hydrogens).
Base Pairing
 Base Pairing
➢A simple mnemonic device for remembering
which bases are paired comes from the line
construction of the capital letters used to identify
the bases.
➢A and T are made up of intersecting straight
lines.
➢In contrast, C and G are largely composed of
curved lines.
➢The RNA base uracil corresponds to thymine,
since U follows T in the alphabet.
 DNA structure
➢ DNA is usually a double-helix and has two
strands running in opposite directions.
➢ There are some examples of viral DNA which
are single-stranded.
➢ Each chain is a polymer of subunits called
nucleotides (hence the name polynucleotide).
➢ Each strand has a backbone made up of (deoxy-
ribose) sugar molecules linked together by
phosphate groups.
 DNA structure

➢ The 3' C of a sugar molecule is connected

through a phosphate group to the 5' C of the
next sugar.
➢ This linkage is also called 3'-5' phosphodiester
linkage.
➢ All DNA strands are read from the 5' to the 3'
end where the 5' end terminates in a phosphate
group and the 3' end terminates in a sugar
molecule.
 DNA structure

➢ Each sugar molecule is covalently linked to one

of 4 possible bases (Adenine, Guanine, Cytosine
and Thymine).
➢ A and G are double-ringed larger molecules
(called purines); C and T are single-ringed
smaller molecules (called pyrimidines).
DNA structure
 DNA structure
➢ In the double-stranded DNA, the two strands
run in opposite directions and the bases pair up
such that A always pairs with T and G always
pairs with C.
➢ The A-T base-pair has 2 hydrogen bonds and
the G-C base-pair has 3 hydrogen bonds.
➢ The G-C interaction is therefore stronger (by
about 30%) than A-T, and A-T rich regions of
DNA are more prone to thermal fluctuations.
➢ The bases are oriented perpendicular to the
helix axis.
 DNA structure

➢ They are hydrophobic in the direction

perpendicular to the plane of the bases (cannot
form hydrogen bonds with water).
➢ The interaction energy between two bases in a
double-helical structure is therefore a
combination of hydrogen-bonding between
complementary bases, and hydrophobic
interactions between the neighboring stacks of
base-pairs.
 DNA structure

➢ Even in the single-stranded state, the bases

prefer to be stacked (like the steps of a spiral
staircase if the bases are identical) and a single-
stranded chain can also have regions of helical
conformation.
 DNA structure

➢ The backbone of polynucleotides are highly

charged (1 unit negative charge for each
phosphate group; 2 negative charges per base-
pair).
➢ If there is no salt in the surrounding medium,
there is a strong repulsion between the two
strands and they will fall apart.
➢ Therefore counter-ions are essential for the
double-helical structure.
 DNA structure

➢ Counter-ions shield the charges on the sugar-

phosphate backbone.
➢ They may also contribute to an attractive
interaction from fluctuating counter-ions
around the backbone, similar to the Van der
Waals interactions for fluctuating induced
dipoles.
 DNA structure
➢The most common DNA structure in solution is
the B-DNA. Under conditions of applied force or
twists in the DNA, or under low hydration
conditions, it can adopt several helical
conformations, referred to as the A-DNA, Z-DNA,
S-DNA...
➢Shown in picture above are three crystallized
states of DNA, the A-DNA (left), B-DNA (middle)
and Z-DNA (right). The A-form crystallizes under
low hydration conditions and is not normally
found for DNA in the cell.
 DNA structure

➢It is, however, the structure adopted by double-

stranded regions in RNA as well as the transient
double-helix between DNA and RNA during
transcription. Both A- and B-DNA are right-
handed helices whereas Z-DNA is a left-handed
helix and is commonly found in regions of DNA
that have an alternating purine-pyrimidine (e.g.
5'-CGCGCGCG-3' or 5'-CGCGCATGC-3')
sequences.
 DNA structure
The table below summarizes some of the major differences.

A-DNA B-DNA Z-DNA

Right-handed helix Right-handed Left-handed

Short and broad Long and thin Longer and

thinner

Helix Diameter 25.5A 23.7A 18.4A

Rise / base-pair 2.3A 3.4A 3.8A

Base-pair / helical turn ~ 11 ~ 10 ~ 12

Helix pitch 25A 34A 47A

Tilt of the bases 20 deg -1 deg -9 deg

 DNA structure

➢ The ball-and-stick representation shown above

can be misleading because it suggests that there
is empty space between the two strands and
between the base-pair stacks. Another
representation is the filled space representation
in which each of the atoms are shown as a ball of
radius representative of its Van der waals
radius. The picture below shows this view for
the 3 DNA structures shown above.
 DNA structure

➢ Here, the B-DNA is on the left and the A-DNA is

in the middle. The blue and white atoms are the
sugar-phosphate backbone atoms, the red are G-
C base-pairs and the yellow are A-T base-pairs.
The B-DNA picture shows very clearly the
'grooves' in between the backbones that also
spiral around the DNA structure; the grooves in
B-DNA come in two sizes, the minor groove and
the major groove.
 DNA structure

➢ A DNA molecule is not a rigid, static structure as

x-ray diffraction pictures might suggest, and the
crystallographic parameters shown above are
average parameters. In reality, each of these
structures are under constant thermal
fluctuations, which result in local twisting,
stretching, bending, and unwinding of the
double-strands. Also, certain sequences lead to
permanent bends or kinks in the direction of the
helix.
 DNA structure

➢ These local (sequence-specific) fluctuations are

essential for the recognition of specific binding
sites along the DNA molecule where proteins
involved in replication, transcription, regulation
of gene expression, or DNA-damage repair can
bind.
 DNA Replication

➢ Before a cell divides, it must make a copy of its

DNA so that both parent and daughter cell have
a complete copy of genetic information.
➢ This process of copying the double-stranded
DNA molecule is called replication.
➢ This process takes place in the nucleus of
eukaryotic cells and the nucleoid region in
prokaryotes.
 DNA Replication

➢ We know that the genetic information of a cell is

contained in the DNA of that cell.
➢ For the cell to divide and produce daughter cells
in mitosis and meiosis it is essential that the
DNA is copied (replicated) and an identical copy
is passed to the daughter cell.
➢ DNA is replicated during interphase of both
mitosis and meiosis.
The assumptions on which the mechanism of DNA
replication are built are:

(i) the evidence that the genetic information was

contained within the nucleic acids;
(ii) the fact that most DNA consists of two strands
of polynucleotide chains, each of which consists
of deoxyribonucleotide residues joined by 3'-5'
phosphodiester bonds; and
(iii) the fact that H-bonds occur between the bases
in the two strands, adenine in one strand is
always bonded to thymine in the other and
likewise cytosine with guanine.
 DNA Replication

Basically, there are five theoretically possible

modes of replication.
1. Conservative
2. Semi-conservative
3. Non-conservative
4. Dispersive
5. End-to-end
 DNA Replication

Conservative
By this mechanism one daughter cell receives the
original DNA molecule whilst the other receives a
completely new copy.
 DNA Replication

Semi-conservative
Here the original DNA molecule is split into two
strands and each strand acts as a template on
which a new strand is synthesized.
 DNA Replication

Non-conservative
Here the original DNA is destroyed completely
during the course of synthesis of two new identical
DNA molecules.
 DNA Replication

Dispersive
Here the original DNA molecule is dispersed or
distributed into all nascent chains.
 DNA Replication

End-to-end
Here the original DNA molecule is present as half
of one chain and the other half of the
complementary chain for both nascent molecules.
The five possible modes of replication are shown schematically
in the following figure.
 DNA Replication

➢ In fact it has been proved that DNA replicates

semi-conservatively, first in E. coli, and
subsequently in all higher organisms as well: it
has been shown to be the case for eukaryotic
cells, including human cells, in tissue culture.
➢ The original experiment which proved this
mechanism was carried out by Meselson and
Stahl in 1958.
The Meselson and Stahl experiment

(a) Meselson and Stahl grew E. coli in a medium

containing 15NH4Cl (i.e. 15N is the heavy isotope
of nitrogen).
The result of this was that 15N was incorporated
into newly synthesized DNA (as well as other
nitrogen-containing compounds).
The Meselson and Stahl experiment

(b) They allowed the cells to grow for many

generations in the 15NH4Cl so that all the DNA
was ‘heavy’ — that is, had a greater density
than normal.
This ‘heavy’ DNA could be distinguished from the
normal DNA by centrifugation in a caesium
chloride (CsCl) density gradient.
Note that 15N is not a radioactive isotope.
The Meselson and Stahl experiment

(c) They then transferred the cells into a medium

with normal 14NH4Cl and took samples at
various definite time intervals as the cells
multiplied, and extracted the DNA that
remained as double-stranded helices.
The Meselson and Stahl experiment

➢ The various samples were then run

independently on CsCl gradients to measure the
densities of DNA present.
➢ The results of this experiment were as follows:
The Meselson and Stahl experiment
The Meselson and Stahl experiment

Thus the DNA that was extracted from the culture

one generation after the transfer from 15N to 14N
medium (i.e. after 20 minutes) had a hybrid or
intermediate density, exactly half-way between that
of DNA containing only 15N and that containing
only 14N; each molecule must therefore have
contained one old and one new strand.
The Meselson and Stahl experiment
➢ DNA extracted from the culture after another
generation (i.e. after 40 minutes) was composed of
equal amounts of this hybrid DNA and of ‘light’
DNA.
➢ As time went on, more and more ‘light’ DNA
accumulated.
➢ The results of the Meselson and Stahl experiment
are not consistent with non-conservative (only 14N-
DNA bands would be expected from 20 minutes
onwards) or conservative (one band of 14N-DNA and
one of 15N-DNA at 20 minutes would be expected)
modes of replication.
The Meselson and Stahl experiment

➢ Thus far, however, the data are consistent with

the three remaining modes of replication.
➢ To distinguish between these, and hence
determine the mode of replication, Meselson
and Stahl carried out an additional experiment
in which they took the DNA isolated at 20
minutes after transfer to 14N-medium, strand
separated this and ran the resultant sample on a
CsCl gradient.
The Meselson and Stahl experiment

➢ For dispersive or end-to-end models we would

predict a single hybrid band, whereas for semi-
conservative repli-cation we would expect two
bands, one light, one heavy.
➢ The latter was found to be the case and hence
DNA replication is a semi-conservative process.
 Mechanism of replication
➢ Watson and Crick, in their 1953 paper in which
they proposed the double-helical structure for
DNA, conclude as follows:
“It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material”.
➢ The implication was that, owing to the strict
complementary nature of the strands, the base
sequence of one strand would determine the
base sequence of the other.
Replication could follow a scheme as shown below.
 Mechanism of replication
➢ The problem, however, with this scheme is that
the energy required for the first step, namely
strand separation, would be far too high to be
plausible.
➢ This led to the suggestion that unfolding took
place a little at a time, so forming a replication
fork as shown.
 Mechanism of replication
➢ Here, as elsewhere in biochemical reactions, an
enzyme is used as a catalyst.
➢ Enzymes lower the energy needed to activate a
reaction, making it more likely to occur.
➢ For E. coli this is an enzyme called DNA poly-
merase III.
➢ The simplest scheme would be for polymerase
molecules to attach themselves to the ends of the
single-stranded DNA template and to synthesize
a complimentary strand of each.
 Mechanism of replication
➢ The first problem encountered with this
proposal arose because the polymerase enzyme
was found to operate in one direction only, that
is it synthesized the nascent DNA strand in the
direction 5'- 3'.
➢ This meant that one of the parent strands
(called the leading strand) could be copied in a
continuous fashion, whereas the other (called
the lagging strand) could only be copied in a
discontinuous way as a series of short lengths
(approx. 1 000 bases long) of DNA, each of
which is called an Okazaki fragment.
Mechanism of replication
 Mechanism of replication

➢ It was also discovered early on that the DNA

polymerase enzyme could not actually
synthesize a DNA molecule from individual
DNA nucleotides but instead required a short
stretch of existing DNA helix duplex on which to
build.
➢ In this sense it is truly a DNA-dependent DNA
polymerase III.
 Mechanism of replication
➢ The full details of the process of replication are
beyond the scope of the A-level syllabus but it
may be of interest to know the roles of some of
the major enzymes/proteins involved in the
process.
➢ The details required for A-level are given in
bold in following table.
➢ Exposure to ionizing radiation and carcinogenic
chemicals, e.g. phenols in cigarette tar, probably
‘overwhelm’ DNA-repair enzymes resulting in
gene mutation and cancerous cells.
Enzymes involved in DNA replication