NUCLEIC ACID CHEMISTRY
NUCLEIC ACID
• Condensation polymers of nucleotides
• They are responsible for the storage and passage of the
information needed for the production of proteins.
• The two structural forms
• Deoxyribonucleic Acid (DNA)
• Ribonucleic Acid (RNA)
• It is composed of a long sugar-phosphate backbone with
organic bases attached to the sugar residues
• The basic repeat unit shown above is known as a Nucleotide
NITROGENOUS BASES
• The nitrogenous bases found in nucleic acids are related
either to the purine ring system or to the pyrimidine ring
system
• Pyrimidine
• Purine
• There are three major pyrimidine bases present in the
nucleotides of both prokaryotes and eukaryotes.
In natural materials, unusual pyrimidine bases occur, e.g
• Bacteria and viral DNA contains 5-methyl cytosine
• Bacteriophages contains 5-hydroxymethyl cytosine
• The two major purine found in living organisms
• Two other purine bases hypoxanthine and xanthine occur as
intermediates in the metabolism of adenine and guanine
• Xanthine is formed by the hydrolysis of guanine and by the
oxidation of hypoxanthine while hypoxanthine is formed by
the hydrolysis of adenine.
• Purine bases containing methyl substituent occur in
foodstuffs and some having pharmacologic properties. E.g.
• Coffee which contains caffeine (1,3,7-trimethylxanthine),
• Tea which contains theophylline (1, 3-dimethylxanthine)
• Cocoa which contains theobromine (3, 7-dimethylxanthine).
• All these are methylxanthines occurring in foodstuffs.
Sugar
• The five carbon monosaccharides (pentoses) are found in
the nucleic acid structure.
• RNA contains D-ribose while DNA contains 2-deoxyribose.
NUCLEOSIDES
• A nucleoside is composed of a purine or a pyrimidine base to
which a sugar (Usually D-ribose or 2-deoxyribose) is
attached.
• The base to sugar linkage is known as β-N-glycosidic linkage
• Examples of nucleosides are the ribonucleosides and 2’-
deoxyribonucleosides. The ribonucleosides comprises of;
adenosine, guanosine, uridine, cytidine
Nucleotides
• These are nucleosides to which phosphoric acid groups are
attached via the sugar unit by esterification.
Polynucleotide structure
• Polymer made by linking several
nucleotide together
• DNA and RNA are two common
polynucleotides
• It has a sugar phosphate backbone with
a nitrogenous base (Purine or
Pyrimidine) attached to every sugar
molecule
• A phosphodiester bond links one
nucleotide to the next
• Each polynucleotide chain has 3’ and 5’
ends.
• All the pentose sugars are involve in the
linkage except the terminal sugars
whose 3’ and 5’ phosphates are free
Synthetic derivatives
• Heterocyclic ring or sugar
moiety can be altered
• synthetic analogs of purines,
pyrimidines, nucleosides and
nucleotides are formed
• Some of the synthetic analogs
are highly useful in clinical
medicine; in therapy of HIV,
HBV, HBC, herpes simplex
virus, chemotherapy of cancer,
treatment of gout etc.
• Termination of nascent DNA
chain is often employed
• Inhibition of enzyme e.g Xanthine
oxidase in gout
Free Nucleotides of Biological importance
• ATP serves as the major supplier of
energy for intracellular processes. It is
the most abundant free nucleotide in
mammalian cell.
• When linked with vitamins or vitamin
derivatives, nucleotides form portion of
coenzymes like NAD, FAD etc.
• Cyclic nucleotides cAMP and cGMP serve
as second messengers or mediators in
hormonally regulated events and other
biological reactions
• Precursors of Nucleic acids; DNA, RNA
• S-adenosylmethionine; a form of active
methionine, serves widely as a methyl
donor in many diverse methylation
reactions SAM
DNA (Deoxyribonucleic Acid)
• In all living cells, DNA serves to store genetic
information.
• Is a double Helical structure, proposed by
James Watson and Francis crick in 1953
• Consist of two stands wind around one another.
• backbone made of alternating sugar
(deoxyribose) and phosphate groups.
• Attached to each sugar is one of four bases--
adenine (A), cytosine (C), guanine (G), and
thymine (T).
• The two strands are held together by bonds
between the bases; adenine bonds with thymine,
and cytosine bonds with guanine.
• The sequence of the bases along the backbones
serves as instructions for assembling protein
and RNA molecules.
• The two strands are antiparallel, i.e., one, strand
runs in the 5' to 3' direction while the other in
3' to 5' direction.
Complementary base pairing
• (A) Thymine pairs with
adenine by 2 hydrogen bonds
(B) Cytosine pairs with
guanine by 3 hydrogen bonds.
• The stacking interaction also
bring stability to the DNA
helix.
• Hydrophobic and
electrostatic interactions
that depends on the
aromaticity of the bases
and their dipole moment
• Can be inter or intra-
strand
DNA DENATURATION
• This refers to the melting of double stranded DNA to
generate two single strands
• The most important contribution to DNA helix stability is
the stacking of the bases on top of one another
• To denature DNA, the main obstacle to overcome is the
stacking energies and the hydrogen bonds that provide
cohesion between base pairs.
• Stacking energies are less for pyrimidine/purine steps, and
for AT-rich regions.
• There are a variety of ways in which to denature DNA.
• The most common (and oldest) methods used in the laboratory is
simply to heat the DNA to a temperature above its melting
temperature (Tm)
• Melting temperature- the temperature at which 50% of double
stranded DNA is changed to single-stranded DNA.
• The unstacking of the DNA base pairs can be readily monitored
spectrophotometrically.
• DNA absorbs strongly at 260nm, and as the DNA melts, the
absorbance will increase until all of the DNA is melted, and then
remains constant on further heating.
• This is called the ‘hypochromic effect’
• The absorbance of single stranded DNA is usually around 50%
greater than that of the corresponding duplex DNA.)
• The actual Tm of a given piece of DNA will depend on several factors
• The length of the DNA sequence (shorter pieces of DNA will tend to
melt more easily than longer pieces),
• The base composition of the DNA (in general, regions with alternating
pyrimidine/purine steps and AT-rich regions will melt more readily),
• The topological condition of the DNA (e.g., whether it is a closed circle
that is relaxed or supercoiled, or a linear piece, or is heavily nicked),
and
• The composition of the buffer (in terms of the amount of salt and
which ions are present) in low salt concentration, a given DNA will melt
at a lower temperature than in a higher salt concentration
RNA (Ribonucleic Acid)
• An intermediate between DNA and proteins
• Monomer: Nucleotide Polymer: Nucleic Acid
• Sugar: ribose
• Backbone: sugar+phosphate
• Uses uracil instead of thymine
• What will “U” bind with in RNA?
• Single stranded
• Directs the process of translation
Types of RNA
• Three types of cellular RNA have been distinguished:
• Messenger RNA (mRNA) or template RNA
• Ribosomal RNA (rRNA) and
• Transfer RNA (tRNA)
• Ribosomal and transfer RNA comprise about 95-97% of all
RNA.
• All three forms of RNA are made from a DNA template.
• Transfer RNA and messenger RNA are synthesized on DNA
templates of the chromosomes, while ribosomal RNA is
derived from nucleolar DNA
Messenger RNA (mRNA)
• This is the RNA that carries information for protein
synthesis from the DNA (genes) to the sites of protein
formation (ribosomes).
• It consists of only 3 to 5% of the total cellular RNA.
• The molecular weight of an average sized mRNA molecule is
about 500,000, and its sedimentation coefficient is 8S.
• The cell does not contain large quantities of mRNA. This is
because mRNA, unlike other RNAs is constantly undergoing
breakdown. It is broken down to its constituent
ribonucleotides by ribonucleases
Messenger RNA is always single stranded. Although there is a certain
amount of random coiling in extracted mRNA, there is no base
pairing. In fact base pairing in the mRNA strand destroys its
biological activity Since mRNA is transcribed on DNA (genes).
Ribosomal RNA (rRNA)
• Found in the ribosomes.
• It comprises about 80% of the total RNA of the cell. The
base sequence of rRNA is complementary to that of the
region of DNA where it is synthesized.
• Ribosomal RNA consists of a single strand twisted upon
itself in some regions
• At low ionic strength rRNA behaves as a random coil, but
with increasing ionic strength the molecule shows helical
regions produced by base pairing between adenine and uracil
and guanine and cytosine.
• Hence rRNA does not show purine-pyrimidine equality.
• The ribosome consists of proteins and RNA. The 70S ribosome of
prokaryotes consists of a 30S subunit and a 50S subunit. The 30S subunit
contains 16S rRNA, while the 50S subunit contains 23S and 5S rRNA.
• The 80S eukaryote ribosome consists of a 40S and a 60S subunit. In
vertebrates the 40S subunit contains 18S rRNA, while the 60S subunit
contains 28-29S, 5.8S and 5S rRNA. In plants and invertebrates the 40S
subunit contains 16-18S RNA, while the 60S subunit contains 25S and 58
and 5.8S rRNA.
Transfer RNA-tRNA OR Soluble RNA–sRNA
• The second most common RNA in the cell is transfer RNA. (10-
20% of total RNA)
• It is also called soluble RNA because it is too small to be
precipitated by ultracentrifugation at 100,000 g. It constitutes
about 10-20% of the total RNA of the cell.
• Transfer RNA is a relatively small RNA having a molecular weight
of about 25,000 to 30,000 and the sedimentation coefficient of
mature eukaryote tRNA is 3.8S.
• Transfer RNA (tRNA) is an essential component of the
protein synthesis reaction.
• There are at least twenty different kinds of tRNA in the
cell and each one serves as the carrier of a specific amino
acid to the site of translation.
• tRNA's are L-shaped molecules.
• The amino acid is attached to one end and the other end
consists of three anticodon nucleotides. The anticodon pairs
with a codon in messenger RNA (mRNA) ensuring that the
correct amino acid is incorporated into the growing
polypeptide chain.
Central Dogma of Molecular Biology
This describes the flow of genetic information in cells from
DNA to messenger RNA (mRNA) to protein. Genes specify
the synthesis of mRNA molecules, which in turn specify the
sequence of proteins.
