Department of Biochemistry & Molecular Biology

Federal University, Dutsin-ma

COURSE CODE: BCH 202
COURSE TITLE: General Biochemistry II (Nucleic Acids Chemistry)

COURSE DURATION: Two hours per week

History of Nucleic acids

Nucleic acids are the principle genetic materials of all living organisms. They are complex
macromolecule containing carbon, hydrogen, oxygen, nitrogen and phosphorous. Living
organisms contain nucleic acid in the form of deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). Some viruses contain only RNA (viroids) e.g. TMV (tobacco mosaic Virus),
poliomyelitis virus. Some viruses contain DNA only example: bacteriophages, Vaccinia and
Adenoviruses. In bacteria and higher cells both types of nucleic acids are found. The nucleic
acids were first isolated in 1868 by miescher from nuclein of pus cells on hospital bandages. He
called it nuclei. He showed that the nuclein contained phosphorus, carbon, oxygen, hydrogen
and nitrogen. Fischer (1880) identified that the Nuclein contains two types of nitrogenous bases
and they are purines and pyrimidines. Altmann (1889) gave the term nucleic acid to replace
nuclein. The name nucleic acid was given to it due to its acidic property. Levene identified a
five-carbon sugar ribose in 1910. He later discovered that there was another type of nucleic acid,
which contains deoxyribose. Nucleic acid that contains ribose sugar is called the ribonucleic acid
(RNA) and that which contains deoxysugar named as the deoxyribonucleic acid. He also
suggested that phosphorus is present in nucleic acid as a phosphate group. Upon hydrolysis,
Nucleic acids yield 3 components namely phosphoric acid, a pentose sugar and nitrogenous
bases.

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There are two types of nucleic acids, namely deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). Nucleic acids play a central role in storage, expression of genetic information and
protein biosynthesis. All nucleic acids are made up from nucleotide components, which in turn
consist of a base, a sugar, and a phosphate residue. DNA and RNA differ from one another in the
type of the sugar and in one of the bases that they contain.

Nitrogenous bases:

The bases that occur in nucleic acids are aromatic heterocyclic compounds derived from either
pyrimidine or purine. Five of these bases are the main components of nucleic acids in all living
system. The purine bases are adenine (Ade), and guanine (Gua) and the pyrimidine base,
cytosine (Cyt) is present in both RNA and DNA. In contrast, uracil (Ura) is only found in RNA.
In DNA, uracil is replaced by thymine (Thy), the 5-methyl derivative of uracil

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PROPERTIES OF BASES:

The pyrimidine ring system is planar, while the purine system deviates somewhat from planarity
in having a slight pucker between its imidazole and pyrimidine portions. Both are relatively
soluble in water. The aromaticity of the pyrimidine and purine ring systems and the electron rich
nature of their OH and NH2 groups made them with the capacity to undergo keto enol tautomeric
shifts. Compounds that exist in two structural isomeric forms which are mutually
interconvertable and exist in dynamic equilibrium are called tautomers and the phenomenon is
termed tautomerism. Pyrimidines and purines exist as tautomeric pairs.

The keto or imino tautomer is called a lactum whereas the enol or amino form is a lactim. It
plays important role in hydrogen bond formation between bases.

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Another property of pyrimidines and purines is their strong absorbance of ultraviolet (UV) light,
which is also a consequence of the aromaticity of their heterocyclic ring structure.

PHOSPHORIC ACID:

The molecular formula of phosphoric acid is H3PO4. It contains three monovalent hydroxyl
groups and divalent oxygen atom, all linked to a pentavalent phosphorus atoms. Through 3’, 5’
phosphodiester bond, nucleotides linked in Nucleic acids.

PENTOSE SUGAR: 

Five carbon sugars are called pentoses. RNA contains the pentose D-Ribose, while 2-deoxy D-
ribose is found in DNA. In both cases, the pentose is in the five membered ring form known as
furanose: D-ribofuranose for RNA and 2-deoxy D-ribose for DNA. When these ribofuranoses
are found in nucleotides, their atoms numbered as 1’, 2’, and 3’ and so on, to distinguish them
from the ring atoms of the nitrogenous bases. An important property of the pentoses is their
capacity to form esters with phosphoric acid. In this reaction, the OH - groups of the pentose,
especially those at C3 and C5 are involved forming a 3’, 5’-phosphodiester bond between
adjacent pentose residues.

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Nucleosides

Nucleosides are compounds formed when a base is linked to a sugar via glycosidic bond. In
nucleosides, the bond is an N-glycosidic because it connects the anomeric C’1 to N-1 of
pyrimidine or to N-9 of a purine. The conformation of N-glycosidic bond is beta in purines and
pyrimidines. Nucleosides are named by adding the ending –idine to the root name of a
pyrimidine or –osine to the root name of purine. The nucleoside adenosine (A) is formed in this
way from adenine and ribose, for example. The corresponding derivatives of the other bases are
called guanosine (G), uridine (U), thymidine (T) and cytidine (C). When the sugar component is
2-deoxyribose, the product is a deoxyribonucleoside e.g., 2-deoxyadenosine.

BASE   SUGAR   NUCLEOSIDES TRIVALNAME

Adenine + Ribose à Adenine ribonucleoside Adenosine

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 Guanine + Ribose à Guanine ribonucleoside Guanosine
Cytosine + Ribose à Cytosine ribonucleoside Cytidine
Thymin + Ribose à Thymine ribonucleoside Thymidine
e
Uracil + Ribose à Uracil ribonucleoside Uridine
Adenine + Deoxyribose à Adenine deoxy ribonucleoside Deoxyadenosine
Guanine + Deoxyribose à Guanine deoxy ribonucleoside Deoxyguanosine
Cytosine + Deoxyribose à Cytosine deoxy ribonucleoside Deoxycytidine
Thymin + Deoxyribose à Thymine deoxy ribonucleoside Deoxythymidine
e
Uracil + Deoxyribose à Uracil deoxy ribonucleoside deoxyuridine

SYN AND ANTI CONFORMATION OF NUCLEOSIDES:

Nucleosides exist either of two conformations designated as syn and anti, pyrimidine nucleosides
favors anti conformation but purine nucleosides favors either syn or anti conformation.
Nucleosides are much more water-soluble than free bases because of the hydrophilicity of the
sugar moiety.

Syn-Adenosine anti-Adenosine  

Mainly nucleosides serve as component parts of nucleotides. In exception, adenosine functions

as an autocoid or local hormone. Adenosine acts locally on specific cells to cause blood vessels
dilation, smooth muscle contraction, neuronal discharge, neurotransmitter release and influence
fat metabolism. Adenosine is also implicated in sleep regulation. Increased levels of adenosine
in brain cells promote sleepiness. Caffeine promotes wakefulness by blocking the interaction of
extracellular adenosine with its neuronal receptors.

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Recently two nucleoside analogues 3’-azidodeoxythymidine and 2’, 3’-dideoxycytidine have
been therapeutically used for the treatment of AIDS (Acquired immune deficiency syndrome)
patients. They inhibit DNA synthesis in HIV by causing premature chain termination due to its
analogues nature to dTTP and dCTP.

Phosphodiester bond

A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two
5-carbon ring carbohydrates (pentoses) over two ester bonds. Phosphodiester bonds are central to
most life on Earth, as they make up the backbone of the strands of DNA. In DNA and RNA, the
phosphodiester bond is the linkage between the 3' carbon atom of one sugar molecule and the 5'
carbon of another, deoxyribose in DNA and ribose in RNA. Hydrolysis of phosphodiester bonds
can be catalyzed by the action of phosphodiesterases which play an important role in repairing
DNA sequences. In biological systems, the phosphodiester bond between two ribonucleotides
can be broken by alkaline hydrolysis because of the free 2' hydroxyl group.

Nucleotides

Nucleotides result when phosphoric acid is esterified to a sugar hydroxyl group of a nucleoside.
Ribose sugar has three hydroxyl groups at C-2’, C-3’ and C-5’ where as deoxysugar has two

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hydroxyl groups at C-3’ and C-5’. The vast majority of monomeric nucleotides in the cell are
ribonucleotides or deoxyribonucleotides having 5’-phosphate groups. Nucleotides have acidic
properties because; they are referred by names like adenylic acid, guanylic acid etc.

  PHOSPHORIC   NUCLEOTIDES TRIVAL NAME

ACID
NUCLEOSIDE
S
Adenosine + Phosphoric acid à Adenosine Adenylic acid
monophosphate(AMP)
Guanosine + Phosphoric acid à Guanosine Guanylic acid
monophosphate(GMP)
Cytidine + Phosphoric acid à Cytidine Cytidylic acid
monophosphate(CMP)
Uridine + Phosphoric acid à Uridine Uridylic acid
monophosphate(UMP)
Deoxyadenosine + Phosphoric acid à deoxyadenosine Deoxy adenylicacid
monophosphate(dAMP)
Deoxyguanosine + Phosphoric acid à Deoxyguanosine Deoxyguanylic acid
monophosphate(dGMP)
Deoxythymidine + Phosphoric acid à Deoxythymidine Deoxythymidilic
monophosphate(dTMP) acid
deoxycytidine + Phosphoric acid à Deoxycytidine Deoxycytidylicacid
monophosphate(dCMP)

Cyclic nucleotides

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Nucleoside monophosphates in which the phosphoric acid is esterified to two of the available
ribose hydroxyl groups are found in all cells. Formation of two ester linkages with one
phosphate results in cyclic structure example: cAMP and cGMP, they are act as secondary
messenger which in turn act as important regulators of cellular metabolism.

Oligonucleotides, polynucleotides
Phosphoric acid molecules can form acid–anhydride bonds with each other. It is therefore
possible for two nucleotides to be linked via the phosphate residues. This gives rise to
dinucleotides with a phosphoric acid–anhydride structure. This group includes the coenzymes
NADP+ and CoA, as well as the flavin derivative FAD. If the phosphate residue of a nucleotide
react with the 3-OH group of a second nucleotide, the result is a dinucleotide with a phosphoric
acid diester structure. Dinucleotides of this type have a free phosphate residue at the 5, end and a
free OH group at the 3 end. They can therefore be extended with additional mononucleotides by
adding further phosphoric acid diester bonds. This is the way in which oligonucleotides, and
ultimately polynucleotides, are synthesized. Polynucleotides consisting of ribonucleotide
components are called ribonucleic acid (RNA), while those consisting of deoxyribonucleotide
monomers are called deoxyribonucleic acid.

DNA
DNA is a polymer of deoxyribonucleotides and it composed of monomeric units namely
deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxycytidylate (dCMP) and

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deoxythymidylate (dTMP). The monomeric deoxynucleotides (or ribonucleotides) in DNA (or
RNA) are held to gather by 3′, 5′- phosphodiester bridges.

Chargaff′s rule of DNA

composition
Ewin chargaff in late 1940s quantitatively
analyzed the DNA sequences from different
species. He observed that in all species he
studied, DNA had equal number of adenine and
thymine residues (A꞊T) and equal number
guanine and cytosine residues (G≡C). This is known as chargaff′s rule of molar equivalence
between the purines and pyrimidines in DNA structure. The significance of chargaff′s rule was
not immediately realized. The double helical structure of DNA derives its strength from chargaff
′s rule. Single stranded DNA and RNAs are usually not obeyed the chargaff′s rule. However,
double stranded RNA which is genetic materials in certain viruses satisfies chargaff′s rule.

DNA Double Helix

The double helical structure of DNA was proposed by James Watson and Francis Crick in 1953
(Nobel prize, 1962).
The elucidation of DNA structure is considered as a great achievement in the era of modern
biology. The structure of DNA double helix is comparable to a twisted ladder, and the salient
features of Watson - Crick Model of DNA are as follows:
 The DNA is right handed double helix. It consist of two polydeoxyribonucleotide chains
(strands) twisted around each other on a common axis
 The two strands are ant parallel that is one strand runs in the 5′ to 3′ direction while the
other in 3′ to 5′ direction.
 The width (or diameter) of a double helix is 2nm
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 Each turn (pitch) of the helix is 3.4nm with 10 pairs of nucleotides, each pair placed at a
distance of about 0.34nm.
 Each strand of DNA has a hydrophilic deoxyribose phosphate backbone (3′-5′
phosphodiester bonds) on the outside (periphery) of the molecule while the hydrophobic
bases are stacked inside (core).
 The two polynucleotide strands are not identical but complementary to each other due to
base pairing.
 The two strands are held to gather by hydrogen bonds formed by the complementary base
pairs. The A-T pair has two hydrogen bonds while G-C pair has three hydrogen bonds.
The G≡C is stronger by about 50% than A꞊T.
 The hydrogen bonds are formed between purines and pyrimidines only. If two purines
face each other, they would not fit into the allowable space. And two pyrimidines would
be too far to form hydrogen bonds. The only base arrangement possible in DNA structure
from spatial consideration is A-T, T-A, G-C and C-G.
 The complementary base pairing in DNA helix prove Chargaff′s rule. The content of
adenine equals to that of thymine (A꞊T) and guanine equals to that of cytosine.
 The genetic information resides on one of the two strands known as template strand or
sense strand. The double helix has (wide) major grooves and (narrow) minor groove
along the phosphodiester backbone. Protein interacts with DNA at these grooves, without
disrupting the base pairs and double helix.

G≡C A꞊T

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Conformations of DNA double helix

Variation in the conformation of the nucleotides of DNA is associated with Conformational

variants of DNA exists in at least six different forms-A to E and Z. Among these, B, A and Z
forms are important.

A-DNA: A-DNA is one of the many possible double helical structures of DNA. A-DNA is
thought to be one of three biologically active double helical structures along with B- and Z-
DNA. It is a right-handed double helix, contains 11 base pairs per turn and there is a tilting of the
base pairs by 20⁰ away from the central axis and its fairly similar to the more common and well-
known B-DNA form, but with a shorter more compact helical structure. It appears likely that it
occurs only in dehydrated samples of DNA, such as those used in crystallographic experiments,
and possibly is also assumed by DNA-RNA hybrid helices and by regions of double-stranded
RNA

B-DNA: The most common form of DNA is B-DNA. The DNA double helix is a spiral polymer
of nucleic acids, held together by nucleotides which base pair together. In B-DNA, the most
common double helical structure, the double helix is right-handed with about 10–10.5
nucleotides per turn. The double helix structure of DNA contains a major groove and minor
groove, the major groove being wider than the minor groove.

Z-DNA: Z-DNA is one of the many possible double helical structures of DNA. It is a left-
handed double helical structure in which the double helix winds to the left in a zig-zag pattern
(instead of to the right, like the more common B-DNA form). Z-DNA is thought to be one of

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three biologically active double helical structures along with A- and B-DNA. Z-DNA is quite
different from the right-handed forms. Formation of this structure is generally unfavorable,
although certain conditions can promote it; such as alternating purine-pyrimidine sequence,
negative DNA supercoiling or high salt and some cations.

A-form B-form Z-form

Other Types of DNA structure
It is now recognized that beside double helical structure, DNA also exists in certain unusual
structure. It is believed that such structures are important for molecular recognition of DNA by
protein and enzymes. This is in fact needed for the DNA to discharge its function in appropriate
manner. Some selected unusual structures of DNA are:

Bent DNA:
In general, adenine base containing DNA tracts are rigid and strength. Bent confirmation of
DNA occurs when A-tracts are replaced by other base or a collapse of the helix into the minor
groove of A-tract. Bending in DNA structure has also been reported due to photochemical
damage or impairing of bases. Certain anticancer drugs produce bent structure in DNA; such
changed structure can take up proteins that damage the DNA.

Triple-stranded DNA:

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Triple-stranded DNA formation may occur due to additional hydrogen bonds between the bases.
Thus, a thymine can selectively form two hydrogen bonds to the adenine of A-T to form T-A-T.
Likewise, a protonated cytosine can also form two hydrogen bonds with guanine of G-C pairs
that result in C-G-C. The triple helix structure is less stable than double helix. This is due to the
fact that the three negatively charge backbone strands in triple helix result in an increased
electrostatic repulsion.

Four-stranded DNA:
Polynucleotides with very high contents of guanine can form a novel tetrameric structure called
G-quartets. These structures are planar and are connected by Hooogsteen hydrogen bonds.
Antiparallel four-stranded DNA structures are referred to as G-tetraplexes. The end of eukaryotic
chromosomes namely telomeres are rich in guanine, and therefore form G-tetraplexes. In recent
years, telomeres have become the targets for anticancer chemotherapies. G-tetraplexes have been
implicated in the recombination of immunoglobin genes and in dimerization of double-stranded
genomic RNA of the human immunodeficiency virus (HIV).

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DNA denaturation
DNA can be denatured under extreme conditions of temperature or pH, which can disrupt the
hydrogen bonding and hydrophobic interactions between the bases, and result in separation of
the strands. Denatured DNA is less viscous than native double-helical DNA, and the bases
exhibit greater UV absorption. The transition from double-stranded DNA (dsDNA) to single
stranded random coil DNA (ssDNA) is called a helix-coil transition.

The denaturation of double stranded DNA is easily followed spectroscopically. The purine and
pyrimidine bases in DNA absorb UV light maximally at a wavelength of approximately 260 nm.
In double-stranded DNA, however, the absorption is decreased due to base-stacking interactions.
When DNA is denatured, these interactions are disrupted and an increase in absorbance is seen
and this change is called the hyperchromic effect. The extent of the effect can be monitored as a
function of temperature.

RNA
RNA is a polymer of ribonucleotides held to gather by 3′, 5′- phosphodiester bridges’ which is
mostly an adaptor molecule, used to translate between information in DNA and protein
machinery/structure. Although RNA has certain similarities with DNA and they have specific
differences which are as follows:
1. Pentose: The sugar in RNA is ribose in contrast to deoxyribose in DNA
2. Pyrimidine: RNA contains the pyrimidine uracil in place of thymine (in DNA)
3. Single strand: RNA is usually single stranded polynucleotides; however, this strand may fold
at certain places to give a double – stranded structure, if complementary base pairs are in close
proximity.

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4. Chargaff’s rule- not obeyed: Due to the single stranded nature, there is no specific relation
between purine and pyrimidine contents. Thus, guanine content is not equal to cytosine (as in the
case of DNA)
5. Susceptibility to alkali hydrolysis: Alkali can hydrolyzed RNA to 2’, 3’- cyclic diesters, this is
possible due to the presence of a hydroxyl group at 2’ position. DNA cannot be subjected to
alkali hydrolysis due to lack of free hydroxyl group
6. Orcinol colour reaction: RNA can be histologically identified by orcinol colour reaction due to
the presence of ribose.

Types of RNA
 mRNA - a transcription of the information in the DNA. Used to carry the information in
temporary format from the DNA archive to the protein synthetic machinery

 rRNA - This RNA forms the core of a large molecular machine, the ribosome, which is
used to make proteins. The ribosome consists of 3-4 RNA molecules and many proteins
(~100 in eukaryotes). Both RNA and proteins are involved in catalysis

 tRNA - an adaptor molecule. Classic "clover-leaf" shapes (secondary structure) folds up

into a 3D L-shape (tertiary structure). The proper amino acid is attached to the CCA end
by an enzyme specific for the amino acid and for the tRNA. One arm, the anticodon loop,
recognizes the code for an amino acid on the mRNA.

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