BIO 203 Biochemistry I

by
Seyhun YURDUGÜL,Ph.D.

Lecture 8
Nucleic Acids
 Content outline
• Nucleosides and nucleotides
• Synthetic nucleotide analogues
• Polynucleotides
• Thermal properties of DNA
• Electrophoresis
 Introduction

– Nucleotides: one of the most important

metabolites of the cell.
– found primarily as the monomeric units
comprising the major nucleic acids of the cell,
RNA and DNA.
– also are required for numerous other important
functions within the cell.
 These functions include:
• 1. serving as energy stores for future use in
phosphate transfer reactions.
• These reactions are predominantly carried
out by ATP.
• 2. forming a portion of several important
coenzymes:
• such as NAD+, NADP+, FAD and coenzyme
A.
NAD (Nicotinamide adenin dinucleotide)
 These functions include:
• 3. serving as mediators of numerous important
cellular processes:
• such as second messengers in signal transduction
events.
• The predominant second messenger:
• Cyclic AMP (cAMP), a cyclic derivative of AMP
formed from ATP.
• 4. controlling numerous enzymatic reactions
through allosteric effects on enzyme activity.
 These functions include:
• 5. serving as activated intermediates in
numerous biosynthetic reactions:
• include S-adenosylmethionine (S-AdoMet)
involved in methyl transfer reactions,
• as well as the many sugar coupled
nucleotides involved in:
• glycogen and glycoprotein synthesis.
 Nucleoside and nucleotide
structure and nomenclature:

• derivatives of the heterocyclic highly basic,

compounds:
• purine and pyrimidine.
                  
                       

Purine Pyrimidine
 Structure
• It is the chemical basicity of the nucleotides
that has given them the common term
"bases"
• as they are associated with nucleotides
present in DNA and RNA.
 Structure
• In cells: Five major bases.
• The derivatives of purine are called adenine
and guanine,
• and the derivatives of pyrimidine are called
thymine, cytosine and uracil.
• The common abbreviations used for these
five bases are, A, G, T, C and U.
 Base Formulas (Base
substitution:X=H)

Thymine, T
Cytosine, C

Adenine, A
Uracil, U

Guanine, G
 Nucleoside forms
• If X=ribose or deoxyribose
• Cytidine
• Uridine
• Thymidine
• Adenosine
• Guanosine
 Nucleotide forms
• If X=ribose phosphate
• Cytidine monophosphate (CMP)
• Uridine monophosphate (UMP)
• Thymidine monophosphate (TMP)
• Adenosine monophosphate (AMP)
• Guanosine monophosphate (GMP)
 Nucleosides
• The purine and pyrimidine bases in cells:
• linked to carbohydrate and in this form:
• termed as ‘nucleosides’.
• The nucleosides are coupled to D-ribose or 2'-
deoxy-D-ribose;
• through a β-N-glycosidic bond:
• between the anomeric carbon of the ribose
• and the N9 of a purine or N1 of a pyrimidine.
 Nucleotides
• The base:
• can exist in 2 distinct orientations about the N-
glycosidic bond.
• These conformations :
• identified as, syn
• and anti.
• ‘Anti’ conformation predominates in naturally
occurring nucleotides.
Syn and anti conformations

syn-Adenosine anti-Adenosine
 Nucleotides
• found in the cell primarily in their
phosphorylated form.
• The most common site of phosphorylation of
nucleotides found in cells:
• the hydroxyl group attached to the 5'-carbon of
the ribose.
 Nucleotides
• The carbon atoms of the ribose present in
nucleotides:
• designated with a prime (') mark to distinguish
them from the backbone numbering in the
bases.
• Nucleotides can exist in the mono-, di-, or tri-
phosphorylated forms.
 Nucleotides
• given distinct abbreviations to allow easy
identification of their structure,
• and state of phosphorylation.
• The monophosphorylated form of
adenosine (adenosine-5'-monophosphate):
• written as, AMP.
 Nucleotides
• The di- and tri-phosphorylated forms:
written as, ADP and ATP, respectively.
• The use of these abbreviations assumes
that:
• the nucleotide is in the 5'-phosphorylated
form.
 Nucleotides
• The di- and tri-phosphates of nucleotides:
linked by acid anhydride bonds.
• Acid anhydride bonds:
• have a high potential to transfer the
phosphates to other molecules.
• due to this property: the nucleotides that
results in their involvement in group
transfer reactions in the cell.
 Nucleotides
• The nucleotides found in DNA:
• unique from those of RNA in that;
• the ribose exists in the 2'-deoxy form and the
abbreviations of the nucleotides:
• contain a d- designation.
• The monophosphorylated form of adenosine
found in DNA (deoxyadenosine-5'-
monophosphate) is written as dAMP.
 Nucleotides
• The nucleotide uridine:
• never found in DNA and thymine is almost
exclusively found in DNA.
• Thymine: found in tRNAs but not rRNAs
nor mRNAs.
• There are several less common bases found
in DNA and RNA.
 Nucleotides
• The primary modified base in DNA is 5-
methylcytosine.
• A variety of modified bases appear in the
tRNAs.
• Many modified nucleotides:
• encountered outside of the context of DNA
and RNA;
• that serve important biological functions.
 Adenosine derivatives

• The most common adenosine derivative is the cyclic

form,
• 3'-5'-cyclic adenosine monophosphate, cAMP.
• This compound is a very powerful second
messenger,
• involved in passing signal transduction events from
the cell surface to internal proteins,
• e.g. cAMP-dependent protein kinase, PKA
 Adenosine derivatives
 cyclic form of GMP (cGMP) : also found in cells
involved as a second messenger molecule.
 In many cases its' role is to antagonize the effects of
cAMP.
 Formation of cGMP occurs in response to receptor
mediated signals similar to those for activation of
adenylate cyclase.
 However, in this case :
 guanylate cyclase coupled to the receptor.
 Adenosine derivatives
☻The most important cGMP coupled signal transduction
cascade:
☻ photoreception.
☻ in this case activation of rhodopsin (in the rods);
☻ or other opsins (in the cones);
☻ by the absorption of a photon of light;
☻through 11-cis-retinal covalently associated with rhodopsin
and opsins
☻ activates transducin,
☻ which in turn activates a cGMP specific phosphodiesterase:
that hydrolyzes cGMP to GMP.
Synthetic nucleotide analogues

• Many nucleotide analogues :

• chemically synthesized and used for their therapeutic
potential.
• nucleotide analogues : can be utilized to inhibit specific
enzymatic activities.
Synthetic nucleotide analogues

• A large family of analogues are used as anti-

tumor agents:
• because they interfere with the synthesis of
DNA
• and thereby preferentially kill rapidly dividing
cells such as tumor cells.
Synthetic nucleotide analogues

• Some of the nucleotide analogues commonly

used in chemotherapy:
• 6-mercaptopurine,
• 5-fluorouracil,
• 5-iodo-2'-deoxyuridine
• 6-thioguanine.
Synthetic nucleotide analogues

• Each of these compounds :

• disrupts the normal replication process,
• by interfering with the formation of correct
Watson-Crick base-pairing.
Synthetic nucleotide analogues

• Nucleotide analogues also have been targeted for

use as antiviral agents.
• Several analogs are used to interfere with the
replication of HIV:
• such as AZT (azidothymidine),
• and ddI (dideoxyinosine).
Synthetic nucleotide analogues
• Several purine analogs: used to treat gout;
• e.g. most common : allopurinol;
• which resembles hypoxanthine.
• Allopurinol inhibits the activity of xanthine oxidase,
• an enzyme involved in de novo purine biosynthesis.
 Synthetic nucleotide analogues

• Additionally, several nucleotide analogues

are used:
• after organ transplantation
• in order to suppress the immune system,
• and reduce the likelihood of transplant
rejection by the host.
 Polynucleotides

• formed by the condensation of two or more

nucleotides.
• condensation most commonly occurs
between the alcohol of a 5'-phosphate of
one nucleotide;
• and the 3'-hydroxyl of a second,
• with the elimination of H2O, forming a
phosphodiester bond.
 Polynucleotides
• The formation of phosphodiester bonds in
DNA and RNA:
• exhibits directionality.
• The primary structure of DNA and RNA
(the linear arrangement of the nucleotides):
• proceeds in the 5' ----> 3' direction.
 Polynucleotides
• The common representation of the primary
structure of DNA or RNA:
• write the nucleotide sequences from left to
right synonymous with the 5' -----> 3'
direction as shown:
• e.g. 5'-pGpApTpC-3'
 Structure of DNA

• Utilizing X-ray diffraction data, obtained from

crystals of DNA,
• James Watson and Francis Crick proposed a model
for the structure of DNA.
 Structure of DNA

• This model predicted that DNA would exist:

• as a helix of two complementary antiparallel
strands,
• wound around each other in a rightward direction,
• and stabilized by H-bonding between bases in
adjacent strands.
 Structure of DNA
• In the Watson-Crick model,
• the bases are in the interior of the helix
aligned at a nearly 90 degree angle relative
to the axis of the helix.
• Purine bases form hydrogen bonds with
pyrimidines,
• in the crucial phenomenon of base pairing.
 Structure of DNA
• Experimental determination has shown that,
in any given molecule of DNA,
• the concentration of adenine (A) is equal to
thymine (T)
 Structure of DNA
• and the concentration of cytidine (C) is
equal to guanine (G).
• This means that A will only base-pair with
T;
• and C with G.
 Structure of DNA
• According to this pattern, known as Watson-
Crick base-pairing:
• the base-pairs composed of G and C contain
three H-bonds,
• whereas those of A and T contain two H-
bonds.
• This makes G-C base pairs more stable than
A-T base-pairs.
A-T Base Pair

G-C Base Pair

 Structure of DNA

• The antiparallel nature of the helix:

• stems from the orientation of the individual
strands.
• From any fixed position in the helix,
• one strand is oriented in the 5' ---> 3'
direction
• and the other in the 3' ---> 5' direction.
 Structure of DNA
• On its exterior surface,
• the double helix of DNA contains two deep
grooves:
• between the ribose-phosphate chains.
• These two grooves are of unequal size;
• and termed the major and minor grooves.
 Structure of DNA
• The difference in their size is:
• due to the asymmetry of the deoxyribose
rings;
• and the structurally distinct nature of the
upper surface of a base-pair;
• relative to the bottom surface.
 Structure of DNA
• The double helix of DNA (A-form)
• has been shown to exist in several different
forms,
• depending upon sequence content and ionic
conditions of crystal preparation.
 Structure of DNA
• The B-form of DNA:
• prevails under physiological conditions of
low ionic strength;
• and a high degree of hydration.
 Structure of DNA
• Regions of the helix that are rich in pCpG
dinucleotides:
• can exist in a novel left-handed helical
conformation termed Z-DNA.
• This conformation results from a 180 degree
change in the orientation of the bases,
• relative to that of the more common A- and B-
DNA.
B-DNA Z-DNA
 Thermal properties of DNA
• As cells divide,
• it is a necessity that the DNA be copied
(replicated),
• in such a way that each daughter cell:
• acquires the same amount of genetic material.
 Thermal properties of DNA
• In order for this process to proceed:
• the two strands of the helix must first be
separated,
• in a process termed ‘denaturation’.
• This process can also be carried out in vitro.
 Thermal properties of DNA
• If a solution of DNA :
• subjected to high temperature, the H-bonds
between bases become unstable;
• and the strands of the helix separate in a
process of thermal denaturation.
 Thermal properties of DNA
• The base composition of DNA:
• varies widely from molecule to molecule
• and even within different regions of the
same molecule.
• Regions of the duplex that have
predominantly A-T base-pairs:
• will be less thermally stable than;
• those rich in G-C base pairs.
 Thermal properties of DNA
• In the process of thermal denaturation,
• a point is reached at which 50% of the DNA
molecule exists as single strands.
• This point is the melting temperature (TM),
• and characteristic of the base composition
of that DNA molecule.
 Thermal properties of DNA
– The TM depends upon several factors in
addition to the base composition.
– These include the chemical nature of the
solvent;
– and the identities and concentrations of ions in
the solution.
 Thermal properties of DNA
• When thermally melted DNA: cooled,
• the complementary strands will again re-form the
correct base pairs,
• in a process termed annealing or hybridization.
• The rate of annealing:
• dependent upon the nucleotide sequence of the
two strands of DNA.
 Analysis of DNA structure
• Chromatography:
• Several of the chromatographic techniques
available for the characterization of
proteins;
• can also be applied to the characterization
of DNA.
• The most commonly used technique:
• HPLC (high performance liquid
chromatography).
 Analysis of DNA structure
• Affinity chromatography technique can be also
employed.
• One common affinity matrix: hydroxyapatite (a
form of calcium phosphate),
• which binds double-stranded DNA;
• with a higher affinity than single-stranded DNA.
 Electrophoresis:
• This procedure:
• serve the same function with regard to DNA
molecules;
• as it does for the analysis of proteins.
• However, since DNA molecules have much
higher molecular weights than proteins,
• the molecular sieve used in electrophoresis
of DNA must be different as well.
 Electrophoresis:
• The material of choice for DNA:
• agarose, a carbohydrate polymer purified
from a salt water algae.
• is a copolymer of mannose and galactose
that when melted and re-cooled:
• forms a gel with pore sizes;
• dependent upon the concentration of
agarose.
 Electrophoresis:
• The phosphate backbone of DNA:
• highly negatively charged,
• therefore DNA will migrate in an electric
field.
• The size of DNA fragments:
• can then be determined by comparing their
migration in the gel to known size
standards.
 Pulsed-field gel electrophoresis

• Extremely large molecules of DNA (in

excess of 106 base pairs):
• effectively separated in agarose gels using
pulsed-field gel electrophoresis (PFGE).
 Pulsed-field gel electrophoresis
• This technique employs two or more electrodes,
• placed orthogonally with respect to the gel,
• that receive short alternating pulses of current.
• PFGE allows whole chromosomes:
• and large portions of chromosomes to be analyzed.
 LITERATURE CITED
• Devlin,T.M. Textbook of Biochemistry with
Clinical Correlations,Fifth Edition,Wiley-Liss
Publications,New York, USA, 2002.
• Lehninger, A. Principles of Biochemistry, Second
edition, Worth Publishers Co., New York, USA,
1993.
• Matthews, C.K. and van Holde, K.E.,
Biochemistry, Second edition, Benjamin /
Cummings Publishing Company Inc., San
Francisco, 1996.