NUCLEIC ACID AND

NUCLEOTIDES
BIOCHEMISTRY

KING EDWARD MEDICAL UNIVERSITY

BATCH 2023
Taimoor Wajid
Biomedical Importance of Nucleotides
1. Precursors of nucleic acids: Without them, DNA or RNA cannot be produced
2. Protein synthesis: Proteins cannot be synthesized or cells cannot proliferate.
3. Energy currency: Nucleotides play an important role as "energy currency" in the cell.
▪ Nucleoside tri- and diphosphates such as ATP and ADP are the principal donors and
acceptors of phosphoryl group in metabolism. By doing this, they play a key role in the
energy transductions
4. Carriers of intermediates: Nucleotides also serve as carriers of activated intermediates in the
synthesis of some carbohydrates, lipids, and proteins.
▪ The sugar derivatives UDP-glucose and UDP-galactose participate in sugar inter
conversions and in the biosynthesis of starch and glycogen
▪ Similarly, nucleoside-lipid derivatives such as CDP-acylglycerol are intermediates in lipid
biosynthesis
5. Co-enzymes: When linked to vitamins nucleotides are structural components of several essential
coenzymes, for example, coenzyme A, FAD, NAD+ and NADP+
6. Regulatory compounds: Nucleotides are important regulatory compounds for many of the pathways
of intermediary metabolism, inhibiting or activating key enzymes.
▪ Roles that nucleotides perform in metabolic regulation include
▪ ATP-dependent phosphorylation of key metabolic enzymes
▪ Allosteric regulation of enzymes by ATP, AMP, and CTP
▪ Control of the rate of oxidative phosphorylation by ADP
7. Nucleotides are used for
▪ Muscle contraction,
▪ Transmission of nerve impulse,
▪ Transports of nutrients across cell membrane
▪ Motility of spermatozoa
8. Sulfate group donor: Adenosine 3'-phosphate-5'-phosphosulfate is the sulfate donor for
▪ sulfated proteoglycans
▪ sulfate conjugates of drugs
9. Methyl group donor: S-adenosylmethionine is a methyl group donor
▪ e.g Nor-adrenaline → Adrenaline by methylation.
10. Second messengers: Nucleotides, such as cyclic AMP (cAMP) and cyclic GMP (cGMP), serve as
second messengers in signal transduction pathways.
11. Signal Transduction: GTP and GDP play key roles in the cascade of events that characterize
signal transduction pathways.
12. Medical applications: Specifically, medical applications include the use of synthetic purine and
pyrimidine analogs that contain halogens, thiols, or additional nitrogen atoms in
▪ The chemotherapy of cancer and AIDS
▪ As suppressors of the immune response during organ transplantation.

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Nucleotide Structure. Nucleotides are composed of:
1. A nitrogenous base (purine or pyrimidine)
2. A pentose monosaccharide
3. One, two, or three phosphate groups
Purines: Both DNA and RNA contain the same purine bases:
▪ Adenine (A) —6-aminopurine
▪ Guanine (G) —2-amino,6-hydroxypurine
Hypoxanthine and xanthine are also purines
The parent nucleotide for purine is Inosine
( Base : Hypoxanthine)
Types of Purine Bases (pu):
Major: (A)&(G)
Minor: Inosine(I) & methyl guanine(7mG)
Unnatural: Mercaptopurine, Allopurinol & 8-Azaguanine
Pyrimidines include:
▪ Cytosine (C)—2-oxy-4-amino-pyrimidine
▪ Thymine (T)—2,4-dioxy-5-methyl-pyrimidine
▪ Uracil (U) —2,4-dioxy-pyrimidine
▪ Both DNA and RNA contain the pyrimidine cytosine (C)
▪ They differ in their second pyrimidine base:
▪ DNA contains thymine (T)
▪ RNA contains uracil (U)
▪ T and U differ by only one methyl
group, which is present on T but
absent on U
▪ The parent pyrimidine is Uracil.
Types of Pyrimidines Bases
• Major: (T), (C) & (U)
• Minor: DHU, 5mC & 5hmC
• Unnatural: Fluorouracil (5FU) & 6-aza cytosine (AZC)
Characteristic of Sugar:
▪ D-family
▪ Aldo pentose
▪ Furanose
▪ β-Anomer
▪ Ribose or deoxy Ribose

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 ▪ Numbered by Prime
▪ Isomerism (2’ endo in DNA, 3’ endo in RNA)

Properties of Nucleotide Bases

1. Aromatic: The Nitrogen containing bases are aromatic i.e. they have alternate double bonds.
2. Heterocyclic: They are heterocyclic i.e. structures that contain, in addition to carbon, other
atoms such as nitrogen
3. Tautomerism: All these bases can exist in keto-enol or
amine-imine form. At physiologic pH keto and amine form is
predominant.
4. Nucleotides Are Polyfunctional Acids: Nucleotides bear significant negative charge at
physiologic pH. The phosphoryl groups can serve as proton donors or acceptors at pH values
approximately two or more units above or below neutrality
5. Weak Bases: Purines or pyrimidines with an –NH2 group are weak bases
6. Functional Groups: The most important functional groups of pyrimidines and purines are
▪ ring nitrogens
▪ carbonyl groups
▪ exocyclic amino groups
7. UV light absorbance: The conjugated double bonds of purine and pyrimidine derivatives
absorb ultraviolet light.
Nucleic acids are characterized by a strong absorption at wavelengths near 260 nm.
The mutagenic effect of ultraviolet light is due to its absorption by nucleotides in DNA
that results in chemical modifications
8. Hydrophobicity: The purine and pyrimidine bases are hydrophobic and relatively insoluble in
water at the near-neutral pH of the cell.
9. Stacking Interaction: Hydrophobic stacking
interactions in which two or more bases are
positioned with the planes of their rings parallel to
each other (like a stack of coins) are one of two
important modes of interaction between bases in
nucleic acids.
Base stacking helps to minimize contact of
the bases with water, and these interactions are
very important in stabilizing the three-dimensional structure of nucleic acids.

10.Note that the smaller pyrimidine molecule has the longer name and the larger purine
molecule the shorter name

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11.Their six-atom rings are numbered in opposite
directions
Unusual Bases
▪ The presence of an unusual base in a
nucleotide sequence may
▪ aid in its recognition by specific
enzymes
▪ protect it from being degraded by
nucleases
Examples of Unusual Bases:
▪ 5-methylcytosine of bacterial and human
DNA
▪ 5-hydroxymethylcytosine of bacterial and
viral nucleic acids
▪ Mono- and di-N-methylated adenine and
guanine of mammalian messenger RNAs that
function in oligonucleotide recognition and in
regulating the half-lives of RNAs

▪ Methylated heterocycles of plants include

▪ Xanthine derivatives caffeine of
coffee
▪ Theophylline of tea e.g
(Dimethylxanthine is a diuretic,
smooth muscle relaxant)
▪ Theobromine of cocoa

▪ Caffeine is a central nervous

system (CNS) stimulant of
the methylxanthine class. It is the world's
most widely consumed psychoactive drug.

▪ Ribose is attach to uracil at carbon no 5 instead of carbon no.1 to

form unusual base pseudouridine (Ψ)

▪ Another unusual base in RNA is TMP (thymidine monophosphate):

Methylation by S-adenosylmethionine of a UMP of preformed on

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 tRNA forms TMP (thymidine monophosphate), which contains ribose rather than
deoxyribose.
Nucleosides
▪ The addition of a pentose sugar to a base produces a nucleoside
▪ If the sugar is D-ribose, a ribonucleoside is produced
▪ If the sugar is 2-deoxy D-ribose, a deoxyribonucleoside is produced
▪ The ribonucleosides of A, G, C, and U are named adenosine, guanosine, cytidine, and
uridine, respectively.
▪ The deoxyribonucleoside of A, G, C, and T have the added prefix, "deoxy-", for example
deoxyadenosine.
▪ The compound deoxythymidine is often simply called thymidine, with the "deoxy"
prefix being understood.
Numbering of Carbon and Nitrogen Atoms
▪ The carbon and nitrogen atoms in the rings of the base
and the sugar are numbered separately
▪ The atoms in the rings of the bases are numbered
▪ 1 to 6 in pyrimidines and
▪ 1 to 9 in purines,
▪ In the pentoses of nucleotides and nucleosides the
carbon numbers are given a prim(´ ) designation to
distinguish them from the numbered atoms of the
nitrogenous base
▪ whereas the carbons in the pentose are numbered 1'
to 5'
▪ Numerals with a prime (e.g., 2' or 3') distinguish atoms
of the sugar from those of the heterocycle.
▪ Thus, when the 5'-carbon of a nucleoside (or nucleotide) is referred to, a carbon atom in
the pentose, rather than an atom in the base, is being specified.
▪ Sugars are linked to the heterocycle by a β -N-glycosidic bond, almost always to the
▪ N-1 of a pyrimidine
▪ N-9 of a purine
▪ The N-glycosyl bond is formed by removal of the elements of water (a hydroxyl group
from the pentose and hydrogen from the base)
Nucleotides
▪ Nucleotides are monophosphate, diphosphate, or triphosphate esters of nucleosides.

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 ▪ The first phosphate group is attached by an ester linkage to the 5’-OH of the pentose.
Such a compound is called a nucleoside 5'-phosphate or a 5'-nucleotide.
▪ Nucleotides are monophosphate, diphosphate, or triphosphate esters of nucleosides.
▪ The first phosphate group is attached by an ester linkage to the 5’-OH of the pentose.
Such a compound is called a nucleoside 5'-phosphate or a 5'-nucleotide.
▪ If one phosphate group is attached to the 5'-carbon of the pentose, the structure is a
nucleoside monophosphate (NMP), like AMP or CMP.
▪ If a second or third phosphate is added to the nucleoside, Nucleoside diphosphate (e.g.,
ADP),Triphosphate (e.g., ATP) results.
▪ The second and third phosphates are each connected to the nucleotide by an acid
anhydride bond which is a "high-energy" bond.
▪ The phosphate groups are responsible for the negative charges associated with
nucleotides, and cause DNA and RNA to be referred to as "nucleic acids."

Mononucleotides
▪ If a phosphate group is added to a nucleoside, forming an ester linkage with the
hydroxyl group of the pentose sugar, a mononucleotide is formed e.g. AMP
▪ In case of ribose there are three such places at carbon atoms
number 2,3 and 5 where an ester linkage can be formed which
are written as
▪ nucleoside 2´-monophosphate
▪ nucleoside 3´-monophosphate
▪ nucleoside 5´-monophosphate respectively
▪ The first phosphate group is attached to the 5´-OH of the
pentose and is called;
▪ nucleoside 5´-monophosphate (NMP) or
▪ 5´-nucleotide e.g Adenosine monophosphate (AMP),

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 ▪ also called adenylate
▪ In contrast to ribose which has three OH groups and thus three points for phosphate
group attachment, deoxyribose has two such places (3´and 5´) as C2 lacks an oxygen
atom and thus named as deoxyribose.
▪ The letter ‘d’ is placed before the name of the nucleotide containing deoxyribose e.g.
dAMP.
Cyclic nucleotides
▪ There are two important cyclic nucleotides:
▪ cAMP
▪ cGMP
▪ cAMP :
▪ Cyclic AMP is a cyclic nucleotide and chemically it is 3´-5´ adenosine
monopohosphate.
▪ It is synthesized in tissues from ATP.
▪ The intracellular cAMP concentration (about 1 nmol/L) is three orders of
magnitude below that of ATP
Functions of c-AMP
▪ Acts as second messenger in the cell
▪ It has role in glycogen metabolism
▪ ↑cAMP, ↑ glycogenolysis
▪ Triglyceride metabolism
▪ ↑ lipolysis
▪ It decreases cholesterol synthesis
▪ It causes activation of protein kinases
▪ It has role in protein biosynthesis
▪ It has role in cell differentiation
▪ It regulates the cell membrane permeability, by increasing permeability of cell
membrane to H2O, Na+, K+ & Ca+2
▪ It modulates both transcription & translation
▪ It regulates insulin secretion, catacholamine biosynthesis & Melatonin synthesis
▪ Histamine ↑ cAMP production in parietal cells which in turn ↑ gastric secretion
▪ Addition of cAMP to malignant cell line in vitro↓ growth rates & restores their
morphology to normal
CGMP
▪ cGMP is sythesized from GTP
Functions of c-GMP:

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 ▪ It serves as a second messenger in response to nitric oxide (NO) during relaxation of
smooth muscle, so it has role in smooth muscle relaxation and vasodilatation
▪ It has role in
▪ Protein phosphorylation
▪ Neurotransmission
▪ Insulin action
▪ Regulation of sodium channels
Nucleoside diphosphates
▪ If a second phosphate is added to the nucleotide, a nucleoside diphosphate is formed
▪ Examples:
▪ Adenosine diphosphate(ADP)
▪ Uridine diphosphate (UDP).
ADP
▪ ADP acts as phosphate acceptor in oxidative phosphorylation
▪ It serve as major biologic transducer of free energy
▪ It has role in
▪ cellular respiration
▪ muscle contraction
▪ enzyme activation
UDP
▪ UDP Glucose is formed by the reaction of UTP with glucose-1 phosphate, which
provides glucose units to;
▪ Glycogen during glycogen synthesis and
▪ Lactose synthesis
▪ Glucosyl disaccharides
▪ The oligosaccharides of glycoproteins and proteoglycans
▪ UDP-sugar derivatives participate in sugar epimerizations
▪ UDP glucose and UDP galactose can be oxidized to UDP glucoronic acid by NAD
dependent dehydrogenase.
▪ UDP glucoronic acid is used for
▪ conjugation and detoxification of bilirubin, benzoic acid, sterols and drugs
▪ for biosynthesis of hyaluronic acid, heparin
▪ UDP galactronic acid and UDP –L Iduronic acid for synthesis of chondroitin SO4.
Nucleoside triphosphates
▪ If a third phosphate is added to a nucleoside diphosphate or nucleoside triphosphate is
formed

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 ▪ Examples:
▪ Adenosine triphosphate (ATP)
▪ Guanosine triphosphate (GTP)
▪ Cytosine triphosphate (CTP)
ATP
▪ The mean intracellular concentration of ATP, the most abundant free nucleotide in
mammalian cells, is about 1 mmol/L
▪ It is the store house of energy for cells.
▪ It has two high energy bonds and on hydrolysis, each releases energy (7.6 Kcal) and this
energy is then used for:
▪ Muscle contraction
▪ Transmission of nerve impulse
▪ Phosphorylations
▪ Active transport
▪ Synthetic processes
▪ Formation of active methionine and active sulfate
GTP
▪ GTP is necessary for the formation of cyclic AMP
▪ For activation of adenyl cyclase by some hormones
▪ GTP serves as
▪ an allosteric regulator
▪ an energy source for protein synthesis
▪ It has role in
▪ Citric acid cycle
▪ Purine synthesis
▪ Gluconeogenesis
CTP
▪ CTP participates in biosynthesis of
▪ Phosphoglycerides
▪ Sphingomyelin
▪ Other substituted sphingosines

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Dinucleotides
▪ The bond between the nucleotides is the
“phosphodiester bond”
▪ This bond is formed mainly between the
3´OH group of sugar of one nucleotide
and 5´PO4 group of sugar of another
nucleotide creating a phosphdiester
linkage.
▪ This forms a dinucleotide.
Phosphodiester Bonds Link Successive Nucleotides in Nucleic Acids
▪ The backbones of both DNA and RNA are hydrophilic.
▪ The hydroxyl groups of the sugar residues form hydrogen bonds with water.
▪ The phosphate groups, are completely ionized and negatively charged at pH 7, and the
negative charges are generally neutralized by ionic interactions with positive charges
which are present on;
▪ proteins, metal ions, and polyamines.
▪ By definition, the 5´ end lacks a nucleotide at the 5´ position and the 3´ end lacks a
nucleotide at the 3´ position.
▪ A polyamine is an organic compound having two or more primary amino groups –
NH2 as cations.
Oligonucleotide
▪ A short nucleic acid is referred to as an oligonucleotide
▪ The definition of “short” is somewhat arbitrary, but polymers containing 50 or fewer
nucleotides are considered as oligonucleotides
Polynucleotide
▪ When nucleotides are joined together by internucleotide bonds, polynucleotides are
formed
▪ Polynucleotides are synonymous with nucleic acids
▪ Well-studied biological nucleic acid molecules range in size from 21 nucleotides to large
chromosomes (human chromosome 1 is a single molecule that contains 247 million base
pairs)
▪ Thus the covalent backbones of nucleic acids consist of alternating phosphate and
pentose residues,
▪ and the nitrogenous bases may be regarded as side groups joined to the backbone at
regular intervals.
▪ The backbones of both DNA and RNA are hydrophilic.

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 ▪ The hydroxyl groups of the sugar residues form hydrogen bonds with water.
Nucleic Acids
▪ There are two types of nucleic acids
▪ DNA
▪ RNA
▪ DNA is present in
▪ Chromosomes in nucleus of eukaryotic organism
▪ Mitochondria and chloroplasts of plants
▪ Prokaryotic cells as plasmids
DNA
▪ DNA is a polymer of deoxyribonucleoside monophosphates covalently linked by 3’ 5’-
phosphodiester bonds.
▪ DNA is a repository of genetic information
▪ In eukaryotic cells, DNA is present in the chromosomes in the nucleus.
▪ It is found associated with basic proteins HISTONES and also various other proteins
present in nucleus (nucleoproteins)
▪ Prokaryotic cells lack nuclei, and have a single chromosome.
▪ The protein-DNA complex is present in a non membrane bound region known as
nucleoid.
▪ It also contain non-chromosomal DNA in the form of plasmids
The properties of Nucleotide bases that affect the structure of Nucleic Acids
▪ The purine and pyrimidine bases are hydrophobic and relatively insoluble in water at
the near-neutral pH of the cell.
▪ Hydrophobic stacking interactions in which two or more bases are positioned with the
planes of their rings parallel (like a stack of coins) are one of two important modes of
interaction between bases in nucleic acids.
▪ The stacking also involves a combination of van der Waals and dipole-dipole interactions
between the bases.
▪ Base stacking helps to minimize contact of the bases with water, and base-stacking
interactions are very important in stabilizing the three-dimensional structure of nucleic
acids.

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 ▪ The most important functional groups of pyrimidines and purines are ring nitrogens,
carbonyl groups, and exocyclic amino
groups.
▪ Hydrogen bonds involving the amino
and carbonyl groups are the second
important mode of interaction
between bases in nucleic acid
molecules. Hydrogen bonds between
bases permit a complementary
association of two (and occasionally
three or four) strands of nucleic acid.
▪ The most important hydrogen-
bonding patterns are those defined by
James D. Watson and Francis Crick in 1953, in which A bonds specifically to T (or U) and
G bonds to C.
▪ These two types of base pairs predominate in double-stranded DNA and RNA.
Nucleic Acid Structure
▪ Nucleic acid structure can be described in terms of hierarchical levels of complexity
(primary, secondary, tertiary)
▪ The primary structure of a nucleic acid is its covalent structure and nucleotide sequence
▪ Any regular, stable structure taken up by some or all of the nucleotides in a nucleic acid
can be referred to as secondary structure
▪ The complex folding of large chromosomes within eukaryotic chromatin and bacterial
nucleoids is generally considered tertiary structure
DNA / Different ways to show primary structure
▪ The nucleotide sequences of nucleic
acids can be represented schematically,
as shown:
▪ The phosphate groups are symbolized
by P , and each deoxyribose is
symbolized by a vertical line, from C-1´
at the top to C-5´ at the bottom (but keep in
mind that the sugar is always in its closed-ring
-furanose form in nucleic acids).
▪ The connecting lines between nucleotides
(which pass through P ) are drawn diagonally
from the middle (C-3´) of the deoxyribose of one nucleotide to the bottom (C-5´) of the next

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 ▪ By convention, the structure of a single strand of nucleic
acid is always written with the 5´ end at the left and the 3´
end at the right—that is, in the 5´ 3´ direction.
▪ Some simpler representations of this penta deoxy
ribonucleotide are pA-C-G-T-A OH, pApCpGpTpA, and
finally 5´-ACGTA-3´.
Primary Structure of DNA
▪ 3’ 5’-Phosphodiester bonds (Sugar-Phosphate Bond)
▪ Successive nucleotides of both DNA and RNA are covalently
linked by PO4 bridges.
▪ Phosphodiester bonds join the 3’-OH group of
deoxypentose of one nucleotide to 5’-PO4 group of an
adjacent nucleotide through a phosphate group.
▪ The back bone of the primary structure is the linear strand
made by sugar phosphate residues, linked together, while
the bases project laterally.
▪ This way a long, un-branched chain is formed.
▪ The resulting long, un-branched chain has polarity.
▪ Both 5’-end and 3’-end are free
▪ at 5’-end there is a free phosphate
▪ at 3’-end there is a free OH that are not attached to other nucleotides
▪ Purines and pyrimidines project laterally from the backbone and forms a variable part.
▪ The variable part is concerned with the transport of genetic material.
Secondary Structure of DNA
▪ DNA exist as double stranded molecule (double helix), except few viruses that contain
single stranded molecule
▪ This model was presented by Watson and Crick in 1953

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 ▪ The two polydeoxyribonucleotide strands are coiled
around a common axis called axis of symmetry
▪ The chains are paired in anti-parallel manner, i.e., the
5’-end of one strand is paired with the 3’-end of the
other strand
▪ In the DNA helix
▪ Deoxyribose-phosphate backbone is
hydrophilic and thus it is on the outside of the
molecule
▪ The bases are hydrophobic and are thus
stacked inside the molecule
▪ The overall structure resembles a twisted ladder
Grooves: The spatial relationship between the two strands in
the helix creates
▪ a major (wide) groove
▪ a minor (narrow) groove
▪ These grooves provide access for the binding of
regulatory proteins to their specific recognition
sequences along the DNA
Clinical aspect: Certain anticancer drugs, such as dactinomycin
(actinomycin D), exert their cytotoxic effect by intercalating
into the narrow groove of the DNA double helix, thus
interfering with RNA and DNA synthesis

Template & Non-Template Strand

▪ The 3´-5´strand is called Template strand while 5´-3´ strand is called Non Template
(coding strand).
▪ The term template strand refers to the sequence of DNA that is copied during the
synthesis of mRNA.

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 ▪ The opposite strand (that is, the strand with a base sequence directly corresponding to
the mRNA sequence) is called the Non Template or coding strand or the mRNA-like
strand because the sequence corresponds to the codons that are translated into protein

Base Pairing
▪ The bases of one strand of DNA are paired with the bases of
the second strand, so that
▪ Adenine is always paired with thymine
▪ Cytosine is always paired with guanine
▪ One polynucleotide chain of DNA double helix is always the
complement of other
▪ Given the sequence of bases on one chain, the sequence of
bases on the complementary chain can be determined
Chargaff’s Rules
▪ The specific base pairing of DNA leads to Chargaff’s Rules:
▪ In any sample of double-stranded DNA:
the amount of adenine equals the amount of thymine
the amount of guanine equals the amount of cytosine
the total amount of purines equals the total amount of pyrimidines ; that is, A + G = T+ C.
▪ The base pairs are held together by hydrogen bonds:
❖ two between A and T
❖ three between G and C
▪ These hydrogen bonds, plus the van der Waals and hydrophobic interactions
between the adjacent stacked bases, stabilize the structure of the double helix
▪ The base composition of DNA varies from one species to another, but is same if
isolated from different tissues of same species
▪ The base composition does not change with age, nutritional status and
environment
Structural forms of the double helix
▪ Based on composition and physiological conditions there are three major structural
forms of DNA:
▪ the B form (described by Watson and Crick in 1953)
▪ the A form

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 ▪ the Z form
B-DNA
▪ The B form is a right-handed helix
▪ It has ten residues per 360° turn of the helix
▪ The planes of the bases are perpendicular to the helical axis
▪ Chromosomal DNA is thought to consist primarily of B-DNA
▪ Transitions between the helical forms of DNA may play an important role in regulating
gene expression
A-DNA
▪ The A form is produced by moderately dehydrating the B form, and the helix is wider
▪ It is also a right-handed helix
▪ There are eleven base pairs per turn
▪ The planes of the base pairs are tilted 20° away from the perpendicular to the helical
axis
▪ Major groove is deeper and minor groove is shallower
▪ The conformation found in DNA-RNA hybrids or RNA-RNA double-stranded regions is
probably very close to the A form
Secondary structure of A-DNA
▪ Generated by de- hydration of B-DNA
▪ It is more compact than B-DNA
▪ Has 2 grooves
▪ Has 11bp/turn
▪ Is right handed
▪ Has a pitch per turn of helix 28 Ao
Z-DNA
▪ Z-DNA is a left-handed helix
▪ It contains about twelve base pairs per turn
▪ The deoxyribose-phosphate backbone "zigzags," hence, the name "Z"-DNA
▪ It is longer and thinner
▪ Major groove is barely apparent and minor groove is deeper, narrow and wedge shaped
▪ Stretches of Z-DNA can occur naturally in regions of DNA that have a sequence of
alternating purines and pyrimidines e.g. poly GC
Secondary structure of Z-DNA
▪ Alternative G -C sequences form this zigzag form DNA
▪ Is more narrower than B-DNA
▪ Has one groove

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 ▪ Has 12 bp/turn
▪ Is left handed
▪ The stacked bases are almost perpendicular
▪ Has a pitch per turn of helix 45.6 Ao

Plasmids

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 ▪ In addition, most species of bacteria also contain small, circular, extrachromosomal DNA
molecules called plasmids.
▪ Plasmid DNA carries genetic information, and undergoes
replication that may or may not be synchronized to
chromosomal division.
▪ Plasmids may carry genes that convey antibiotic resistance
to the host bacterium, and may facilitate the transfer of genetic information from one
bacterium to another.

Certain DNA Sequences Adopt Unusual Structures

Palindrome
▪ A rather common type of DNA sequence is a palindrome.
▪ A palindrome is a word, phrase, or
sentence that is spelled identically read
either forward or backward; two examples
are ROTATOR and NURSES RUN.
▪ The term is applied to regions of DNA with
inverted repeats of base sequence having
two fold symmetry over two strands of
DNA.
Hairpin DNA Structure
▪ Such sequences are self complementary within each
strand and therefore have the potential to form
hairpin or cruciform (cross-shaped) structures.
Mirror Repeat
▪ When the inverted repeat occurs within each
individual strand of the DNA, the sequence is called a
mirror repeat
▪ Mirror repeats do not have complementary
sequences within the same strand and cannot
form hairpin or cruciform structures.
▪ Sequences of these types are found in virtually
every large DNA molecule and can encompass a
few base pairs or thousands.

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Separation of the two DNA strands in the double helix
▪ The two strands of the double helix separate when hydrogen bonds between the paired
bases are disrupted. Disruption can occur in the laboratory if

▪ the pH or the salt concentration of the DNA solution is altered so that the
nucleotide bases ionize
▪ if the solution is heated above 80°C (Phosphodiester bonds are not broken by
such treatment)
▪ Disruption of the hydrogen bonds between paired bases and of base stacking causes
▪ Unwinding of the double helix to form two single strands (complete separation
of DNA stands along the entire length)
▪ Part of the length (partial denaturation) of the molecule.
▪ No covalent bonds in the DNA are broken.
Denaturation
▪ Duplexes of two RNA strands can also be denatured.
▪ RNA duplexes are more stable than DNA duplexes.
▪ At neutral pH, denaturation of a double helical RNA often requires temperatures 20 C or
more higher than those required for denaturation of a DNA molecule with a comparable
sequence.
▪ Tm: When DNA is heated, the temperature at which one half of the helical structure is
lost is defined as the melting temperature.
▪ Viral or bacterial DNA molecules in solution denature when they are heated slowly. The
loss of helical structure is called Denaturation.
▪ Each species of DNA has a characteristic
denaturation temperature, or melting point
(tm)
▪ Careful determination of the melting point of a
DNA specimen, under fixed conditions of pH
and ionic strength, can yield an estimate of its
base composition
Factors affecting Tm
▪ The Tm is influenced by
▪ the base composition of the DNA
▪ the salt concentration of the solution
▪ The higher the content of GC base pairs, the higher the melting point of the DNA. This is
because GC base pairs, with three hydrogen bonds, require more heat energy to
dissociate than AT base pairs.

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 ▪ An increase in salt concentration increases and a decrease in salt concentration
decreases the Tm
▪ Denaturation can be monitored by measuring its absorbance at 260 nm
Hyperchromacity of denaturation:
Concomitant with this
denaturation of the DNA molecule is
an increase in the optical absorbance
of the purine and pyrimidine bases—
a phenomenon referred to as
hyperchromicity of denaturation.
▪ So single-stranded DNA has a
higher relative absorbance at
this wavelength than does
double-stranded DNA

Renaturation or Reannealing
▪ Under appropriate conditions (temp. & salt
concentration), separated strands of DNA will
renature or reassociate and form the double helix by
the process called renaturation (or reannealing).
▪ This reannealing process is also often referred to as
hybridization.
▪ When the temperature or pH is returned to the
range in which most organisms live, the unwound
segments of the two strands spontaneously rewind,
or anneal, to yield the intact duplex
▪ Renaturation of a DNA molecule is a rapid one-step process
▪ The rate of reassociation depends upon the concentration of the complementary
strands

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Super coiling In Eukaryotes
▪ Eukaryotic DNA is associated with tightly bound basic proteins, called histones.
▪ These serve to order the DNA into basic
structural units, called Nucleosomes that
resemble beads on a string.
▪ Nucleosomes are further arranged into
increasingly more complex structures that
organize and condense the long DNA
molecules into chromosomes.
▪ Nucleosomes can be packed more tightly
to form a polynucleosome also called a
nucleofilament or a 30-nm fiber.
▪ The fiber is organized into loops that are
anchored by nuclear scaffolding proteins.
▪ Additional levels of organization lead to the final
chromosomal structure
▪ There are five classes of histones, designated H1, H2A, H2B,
H3, and H4.
▪ These small proteins are positively charged at
physiologic pH as a result of their high content of
lysine and arginine.
▪ Because of their positive charge, they form ionic
bonds with negatively charged DNA
▪ Nucleosomes: Two molecules each of H2A, H2B, H3,
and H4 form the structural core of the individual
"beads."
▪ Around this core, a segment of the DNA double helix is wound nearly twice, forming a
negatively super twisted helix.
▪ The N-terminal ends of these histones can be acetylated, methylated, or
phosphorylated.
▪ These reversible modifications can influence how tightly the histones bind to the DNA,
thereby affecting the expression of specific genes.
▪ Neighboring nucleosomes are joined by "linker DNA” approximately 50 base pairs long.
.
▪ Histone H1 of which there are several related species, is not found in the nucleosome
core.
▪ But instead binds to linker DNA chain between the nucleosomes beads

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RNA
▪ The genetic master plan is contained in the nucleotide sequence of
DNA.
▪ It is through the ribonucleic acid (RNA)―the "working copies" of the
(DNA) ― that the master plan is expressed
▪ RNA is a polymer of ribonucleotides of Adenine, Uracil, Guanine and
Cytosine, joined together by 3´-5´phosphodiester bonds.
▪ RNA does not contain thymine except in rare cases.
▪ The pentose sugar of RNA is Ribose.
▪ Location: RNA is found in the nucleolus, ribosomes, mitochondria, and
cytoplasm.
▪ There is also a wide variety of special-function RNAs, including some
(called ribozymes) that have enzymatic activity.
Primary Structure of RNA
▪ It is defined as the number and sequence of ribonucleotides in the RNA chain.
▪ The sequence is complementary to the
template strand of the gene from which it
was transcribed.
▪ The ribonucleotides are held together by 3´
5´ phosphodiester bonds.
▪ 3´-OH group of one nucleotide is bound to
5´-PO4 of the other nucleotide and form a
linear strand.
▪ Phosphodiester bonds are present between
ribosyl moieties (ribose sugar)
▪ The ribosyl moieties are attached to the
nucleobases (nitrogenous bases) by N-glycosidic bonds
▪ The polymer has a polarity
Secondary Structure of RNA
▪ RNA has no simple, regular secondary structure that serves as a
reference point, as does the double helix for DNA.
▪ The three-dimensional structures of many RNAs, like those of proteins,
are complex and unique.
▪ Secondary structure involves coil formation of the polyribonucleotide
chain.
▪ The coiled structures are stabilized by
▪ Hydrophobic interactions between purine and pyrimidine bases.

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 ▪ Intra-chain hydrogen bonds
between G-C and A-U.
▪ Weak interactions,
especially base-stacking
interactions.
▪ Where complementary sequences
are present, the predominant
double-stranded structure is
▪ A-form, a right-handed double
helix.
▪ Z-form helices have been made in the laboratory (under very high-salt or high-
temperature conditions).
▪ The B form of RNA has not been observed.
Tertiary Structure of RNA
▪ This involves the folding of the molecule into three dimensional
structure.
▪ There is cross-linking at various sites stabilized by hydrophobic
and hydrogen bonds producing a compactly coiled globular
structure.
Differences between DNA and RNA
Although sharing many features with DNA, RNA possesses several specific differences:
1. Size: RNA is considerably smaller than DNA
2. Sugar: In RNA, the sugar moiety to which the phosphates and purine and pyrimidine bases
are attached is ribose rather than the 2'-deoxyribose of DNA
3. Pyrimidine: The pyrimidine components of RNA differ from those of DNA. Instead of
thymine, RNA contains the ribonucleotide of uracil. Thymine is present in the rare case of tRNA
4.Single Strand: RNA typically exists as a single strand,
whereas DNA exists as a double-stranded helical molecule.
▪ However, given the proper complementary base sequence with opposite polarity, the
single strand of RNA is capable of folding back on itself like a hairpin and
thus acquiring double-stranded characteristics: G pairing with C, and A with U.
5. G≠C: Since the RNA molecule is a single strand complementary to only one of the two strands
of a gene, its guanine content does not necessarily equal its cytosine content, nor does its
adenine content necessarily equal its uracil content.
6. Hydrolysis: RNA can be hydrolyzed by alkali to 2',3' cyclic diesters of the mononucleotides,
compounds that cannot be formed from alkali-treated DNA because of the absence of a 2'-
hydroxyl group.
7. Location: In addition to nucleus, RNA is found in cytoplasm.

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8. Reverse Transcription: DNA forms RNA by transcription but RNA cannot form DNA. In
experiments, reverse transcriptase can be used for this purpose
Types of RNA
▪ Important classes of RNA
▪ Messenger RNA (mRNA)
▪ Transfer RNA (tRNA)
▪ Ribosomal RNA (rRNA)
▪ Small Nuclear RNA (snRNA)
Messenger RNA ( mRNA )
▪ This class is the most heterogeneous in
▪ abundance
▪ size (500-6000 nucleotides)
▪ base sequence
▪ Stability
▪ mRNA comprise about 2–5% of total cellular RNA
▪ mRNA molecules are formed with the help of DNA template strand (3´-5´) during the
process called transcription.
▪ In addition to the protein coding regions that can be translated, there are untranslated
regions at its 5´ and 3´ ends
Functions of mRNA:
▪ The members of this class function as messengers to convey the information in a gene
to the protein synthesizing machinery (ribosomes).
▪ The mRNA carries genetic information from the nuclear DNA to the cytosol, where it is
used as a template for protein
synthesis.
Polycistronic and Monocistronic
mRNA:
▪ Polycistronic: If the mRNA carries
information from more than one
gene (i.e. it carries the code for more than one polypeptide) it is called polycistronic
which is characteristic of prokaryotes.
▪ Prokaryotes can have both polycistronic and
monocistronic mRNAs.
▪ Monocistronic: If the mRNA carries
information from just one gene (i.e. it carries
the code for only one polypeptide) it is called
monocistronic, a characteristic of eukaryotes.

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Special structural characteristics of eukaryotic messenger RNA
▪ A Cap: There is a “cap” on the 5´ end consisting of a molecule of 7-methylguanosine
(methylated nucleoside) attached "backward" (5’-5’) through a triphosphate linkage
▪ The cap is involved in
▪ The recognition of mRNA by the translation machinery
▪ Stabilizing mRNA against 5´exonucleases
▪ Poly-A tail: It contains a long sequence of adenine nucleotides (20-250) on the 3´-OH
end of the RNA chain and is called “poly-A tail”
▪ It is thought to play role
▪ In stability of
mRNA against
3´
exonucleases
▪ Facilitates
translation
▪ Both the mRNA "cap"
and "poly(A) tail" are
added post-
transcriptionally to
mRNA precursor
molecules (pre mRNA).
Transfer RNA (tRNA)
▪ t RNA is the smallest of the three major species of RNA (4S).
▪ They are single stranded globular molecules.
▪ They remain largely in cytoplasm.
▪ They are generated by nuclear processing of a precursor molecule.
▪ tRNAs compose roughly 20% of total cellular RNA
▪ There are at least 20 species of tRNA molecules in every cell.
▪ Although each specific tRNA differs from the others in its sequence of nucleotides, the
tRNA molecules as a class have many features in common
Primary structure
▪ t RNA molecules consist of 74-95 nucleotides in a particular sequence.
▪ The t RNA molecules contain not only the usual bases like adenine, guanine, cytosine,
uracil but also contain unusual bases like
▪ Dihydrouracil (DHU)
▪ Pseudouridine (uracil attached to 5’ of Ribose)
▪ Thymine (formed by methylation of UMP)

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Secondary Structure
▪ Each single stranded
t RNA is folded extensively.
▪ Extensive intra chain base pairing which leads to a
characteristic CLOVER-LEAF structure.
▪ These folds are stabilized by hydrogen bonds
between complementary bases of the same strand.
Arms or loops of tRNA
All tRNA molecules contain 4 main arms or loops.
1-Acceptor arm: This is made up of unpaired sequences of
cytosine-cytosine-adenine (CCA) at the 3´end.
▪ The 3´OH group of adenine binds with the
carboxylic group of a specific amino acid and carries
it to ribosomes for protein synthesis.

2-Anticodon arm: It is in the form of a loop and carries specific sequences of three bases which
constitute the anticodon.
▪ The bases of anticodon are bonded with three complementary bases of codon of mRNA.
3-D arm: It contains the base dihydrouridine.
4-TΨC arm: It contains thymine, pseudouridine and cytosine.
▪ The extra arm and the TΨC arms help to define a specific tRNA.
Function of tRNA
▪ The t RNA molecules serve as ADAPTERS for the translation of information in the
sequence of nucleotides of the mRNA into specific amino acids.
▪ There is at least one (and often several) specific type of tRNA molecule for each of the
amino acids commonly found in proteins.
▪ Each t RNA carries its specific amino acid to the site of protein synthesis.
▪ There it recognizes the genetic code word on mRNA and this specifies the addition of its
amino acids to the growing peptide chain.
Ribosomal RNA (r RNA)
▪ Ribosomal RNA (r RNA) is found in association with several proteins as a component of
ribososmes--- a cytoplasmic nucleoprotein structure that acts as the machinery for the
synthesis of proteins from the mRNA template.
▪ RNAs make up 80% of the total RNA in the cell.
▪ The ribosomal subunits are defined according to their sedimentation velocity in
Svedberg units.
▪ Svedberg unit is related to the molecular weight and shape of the compound.

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 ▪ The bases in r RNA are mainly adenine, guanine, cytosine and uracil and a few
pseudouridine
Eukaryotic Ribosome
▪ Eukaryotic ribosome: The mammalian ribosome contains two major nucleoprotein
subunits:
▪ a larger one with 60S
▪ a smaller one with 40S.
The 60S subunit contains
▪ a 5S rRNA
▪ a 5.8S rRNA
▪ a 28S rRNA
▪ more than 50 specific
polypeptides.
The 40S subunit is smaller and contains
▪ a single 18S rRNA
▪ Approx. 30 distinct polypeptide
chains.
Prokaryotic Ribosomes
▪ The prokaryotic ribosome consist of
▪ a larger 50S subunit
▪ A smaller 30S subunit
▪ 50 S subunit contains
▪ 5S rRNA
▪ 23S rRNA
▪ 30 S subunit contains a 16S
rRNA
rRNA
▪ In eukaryotes, all of the ribosomal RNA molecules except the 5S rRNA, which is
independently transcribed, are processed from a single 45S precursor RNA molecule in
the nucleolus
▪ The rRNA molecules are packed in the nucleolus with the specific ribosomal proteins.
▪ In the cytoplasm, the ribosomes remain quite stable and capable of many translation
cycles
▪ Functions of rRNA:
▪ The rRNA are necessary for ribosomal assembly and play a key role in the binding of
mRNA to ribosomes and its translation.
▪ Some rRNA function as catalysts in protein synthesis and are termed as “ribozymes”.

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Small nuclear RNA (SnRNA)
▪ Small nuclear RNA (SnRNA) are large number of small stable RNA species found in
eukaryotic cells.
▪ Most of them are complexed with proteins to form ribonucleoproteins.
▪ They are distributed in the nucleus, in the cytoplasm or in both.
▪ They are significantly involved in rRNA and mRNA processing and gene regulation.

Denaturation of RNA
▪ Duplexes of two RNA strands can also be denatured.
▪ RNA duplexes are more stable than DNA duplexes.
▪ At neutral pH, denaturation of a double helical RNA often requires temperatures 20° C
or more higher than those required for denaturation of a DNA molecule with a
comparable sequence.

NUCLEOTIDE ANALOGS
It is possible to alter heterocyclic ring or sugar moiety, and produce synthetic analogs of
purines, pyrimidines, nucleosides and nucleotides. Some of the synthetic analogs are highly
useful in clinical medicine.
The pharmacological applications of certain analogs are listed below
1. Allopurinol is used in the treatment of hyperuricemia and gout

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 2. 5-Fluorouracil, 6-mercaptopurine, 8-aza-guanine, 3-deoxyuridine, 5- or 6-azauridine, 5-
or 6-azacytidine and 5-idouracil are employed in the treatment of cancers. These
compounds get incorporated into DNA and block cell proliferation.
3. Azathioprine (which gets degraded to 6-mercaptopurine) is used to suppress
immunological rejection during transplantation.
4. Arabinosyladenine is used for the treatment of neurological disease, viral encephalitis.
5. Arabinosylcytosine is being used in cancer therapy as it interferes with DNA replication.
6. The drugs employed in the treatment of AIDS namely zidovudine or AZT (3-azido 2c,3c-
dideoxythymidine) and didanosine (dideoxy-inosine) are sugar modified synthetic
nucleotide analogs

University Questions
▪ Compare and contrast the structural and other features of DNA and RNA.
▪ What is the structure of DNA?
▪ Draw the structure of ADENOSINE TRIPHOSPHATE (ATP). Enumerate four functions of
ATP.
▪ (a) Define with one example.
i. NUCLEOSIDE
ii. NUCLEOTIDE
(b) Give four functions of NUCLEOTIDES in the human body.
▪ Name the NITROGENOUS BASES found in RNA. What are the major classes of RNA?
Write down characteristic features of any two classes.
▪ What are the characteristic structural features and functions of mRNA and tRNA?

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