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Nucleic acid chemistry
University of BenghaziFaculty of medicine
Department of Biochemistry
1
History:
Friedrich Miescher in 1869
• isolated what he called nuclein from the nuclei of pus cells
• Nuclein was shown to have acidic properties, hence it became called nucleic acid
2
Two types of nucleic acid are found
• Deoxyribonucleic acid (DNA)• Ribonucleic acid (RNA)
3
The distribution of nucleic acids in the eukaryotic cell DNA is found in the nucleus
with small amounts in mitochondria and chloroplasts RNA is found throughout the cell
4
NUCLEIC ACID STRUCTURE Nucleic acids are polynucleotides Their building blocks are nucleotides
© 2007 Paul Billiet ODWS 5
Definition:
Nucleic acids are polymers containing nitrogenous bases attached to sugar–phosphate backbones. The common nitrogenous bases of nucleic acids are
1-The bicyclic purines, adenine and guanine.
2-The monocyclic pyrimidines, cytosine, uracil, and thymine
6
NUCLEOTIDE STRUCTURE
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PHOSPATE SUGAR
Ribose or Deoxyribose
NUCLEOTIDE
BASEPURINES PYRIMIDINES
Adenine (A)Guanine(G)
Cytocine (C)Thymine (T)Uracil (U)
© 2007 Paul Billiet ODWS
1-Nitrogenous Bases Planar, aromatic, and heterocyclic Derived from purine or pyrimidine Numbering of bases is “unprimed”
8
Aromaticity: Note that all five bases can be considered aromatic: they are cyclic
Often the bases are referred to as ‘heterocycles’. Recall from your organic chemistry that a heterocyclic compound, or heterocycle, is a cyclic compound
in which one or more of the atoms of the ring are hetero atoms
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Purine and pyrimidine bases
10
There are also derivative of purine bases are:
11
Nucleic Acids Also Contain Additional Bases
-Small quantities of additional purines and pyrimidines occur in DNA
and RNAs .
Examples: include 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, these atypical bases function in oligonucleotide
recognition and in regulating the half-lives of RNAs. 12
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2-SugarRibose is a pentose
C1
C5
C4
C3 C2
O
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RIBOSE DEOXYRIBOSE
CH2OH
H
OH
C
C
OH OH
C
O
H HH
C
CH2OH
H
OH
C
C
OH H
C
O
H HH
C
Spot the difference
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SugarsD-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group
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Deoxyribose (like ribose) is a sugar with 5 carbon atoms in a ringOxygen is one of the ring members
In Deoxyribose, one of the OH groups is missing and replaced with hydrogen, Thus deoxy = - 1 oxygen
18
• Nitrogenous bases
• In DNA the four bases are:– Thymine– Adenine– Cytosine– Guanine
• In RNA the four bases are:– Uracil– Adenine– Cytosine– Guanine
Base - pairingSummary of nitrogenous bases
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Nucleosides
N9
C1’
1-Purines bond to the C1’ carbon of the sugar at their N9 atoms2-Pyrimidines bond to the C1’ carbon of the sugar at their N1 atoms
Result from linking one of the sugars with a purine or pyrimidine base through an N-glycosidic linkage
20
3-Phosphate Groups Mono-, di- or triphosphates
Phosphates can be bonded to either C2, C3 or C5 atoms of the sugar
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Definitions:
1. Nucleobase: the base only 2. Nucleoside: base attached to sugar 3. Nucleotide: base, sugar and phosphate. The
phosphate can be attached to the 5’,3’, or 2’ carbon.
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Nucleotide
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Nucleotides
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Deoxyribonucleotides of nucleic acids
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Ribonucleotides of nucleic acids
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Some adenosine monophosphates
28
Physiological importance of nucleotides:
Nucleoside Triphosphates are important energy carriers (ATP, GTP)
Important components of coenzymes:(FAD, NAD+ and Coenzyme A).
Act as a carrier of activated metabolites: (UDP-G & CDP-choline). Act as metabolic regulators and signaling
molecles: (cAMP & cGMP).29
Directionality the sugar-phosphate backbone The nucleotides are all
orientated in the same direction The phosphate group joins the
3rd Carbon of one sugar to the 5th Carbon of the next in line.
P
P
P
P
P
P
© 2007 Paul Billiet ODWS 30
ADDING IN THE BASES
The bases are attached to the 1st Carbon
Their order is important
It determines the genetic information of the molecule
P
P
P
P
P
P
G
C
C
A
T
T© 2007 Paul Billiet ODWS 31
DNA IS MADE OF TWO STRANDS OF POLYNUCLEOTIDE
P
P
P
P
P
P
C
G
G
T
A
A
P
P
P
P
P
P
G
C
C
A
T
T
Hydrogen bonds
© 2007 Paul Billiet ODWS 32
Phosphodiester bonds link successive nucleotides in
nucleic acids
Phosphodiester linkages in the covalent backbone of DNA and RNA
33
Polynucleotides1-The condensation two nucleotides 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 .
2-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.
3-The 1ry structure of DNA or RNA molecules is to write the
nucleotide sequences from left to right with the 5'—>3' direction as: 5'–pGpApTpC–3'
34
. Nucleic Acids DNA –deoxyribonucleic acid
Polymer of deoxyribonucleotide triphosphate (dNTP)
4 types of dNTP (ATP, CTP, TTP, GTP) All made of a base + sugar + triphosphate
RNA –ribonucleic acid Polymer of ribonucleotide triphosphates (NTP) 4 types of NTP (ATP, CTP, UTP, GTP) All made of a base + sugar + triphosphate
So what’s the difference? The sugar (ribose vs. deoxyribose) and one
base (UTP vs. TTP) 35
Nucleotides Absorb Ultraviolet LightThe conjugated double bonds of purine and pyrimidinederivatives absorb ultraviolet light. The mutagenic effectof ultraviolet light results from its absorption bynucleotides in DNA with accompanying chemicalchanges. While spectra are pH-dependent, at pH 7.0 allthe common nucleotides absorb light at a wavelengthclose to 260 nm .
36
SYNTHETIC NUCLEOTIDE ANALOGS ARE USED IN CHEMOTHERAPY
1-Synthetic analogs of purines, pyrimidine, nucleosides, and nucleotides altered in either the heterocyclic ring or the sugar moiety have numerous applications in clinical medicine.
2-Their toxic effects reflect either inhibition of enzymes essential for nucleic acid synthesis or their incorporation into nucleic acids with resulting disruption of base-pairing.
3-Oncologists employ 5-fluoro- or 5-iodouracil, 3-deoxyuridine and 6-thioguanine which are incorporated into DNA prior to cell division..
37
4-The purine analog allopurinol, used in treatment of hyperuricemia and gout, inhibits purine biosynthesis and xanthine oxidase activity.
5-Cytarabine is used in chemotherapy of cancer.
6-Finally, azathioprine, which is catabolized to 6-mercaptopurine,is employed during organ transplantation to suppress immunologic rejection..
38
CLINICAL USAGE SYNTHETIC NUCLEOTIDE ANALOGS
Synthetic nucleotide analogues Clinical application
5-fluoro- or 5-iodouracil
Cancer treatment…incorporated into DNA prior to cell division
Allopurinol Gout treatment
Azathioprine-catabolized to 6-mercaptopurine
suppress immunologic rejection in organ transplantation
3-deoxyuridine and 6-thioguanine Cancer treatment…incorporated into DNA prior to cell division
39
Synthetic purine and pyrimidine analogs
40
DNA IS MADE OF TWO STRANDS OF POLYNUCLEOTIDE The sister strands of the DNA molecule run in opposite
directions (antiparallel) They are joined by the bases Each base is paired with a specific partner:A is always paired with T G is always paired with CPurine with Pyrimidine This the sister strands are complementary but not
identical The bases are joined by hydrogen bonds, individually
weak but collectively strong41
Watson & Crick Base pairing
42
The Double Helix (1953)
© Dr Kalju Kahn USBC Chemistry and BiochemistryPublic Domain image
43
Schematic representation of the nucleotide sequences of nucleic acids
44
Complementarity of strands in the DNA double helix
Watson-Crick model for the structure of DNA
45
CHARGAFF’S RULES:
• Base pairing rule is A-T and G-C
• Thymine is replaced by Uracil in RNA
• Bases are bonded to each other by Hydrogen bonds
• Discovered because of the relative percent of each base; (notice that A-T is similar and C-G are similar)
46
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DNA
•Information for all proteins stored in DNAin the form of chromosomes or plasmids.
•Chromosomes (both circular and linear) consist of two strands of DNA wrapped together in a left handed helix.
•The strands of the helix are held together by hydrogen bonds between the individual bases.
•The “outside” of the helix consists of sugar and phosphate groups, giving the DNA molecule a negative charge.
DNA and RNA are polynucleotides
• Both DNA and RNA are polynucleotides.
• They are made up of smaller units called nucleotides.
• DNA is made of two polynucleotide strands:
• RNA is made of a single polynucleotide strand:
49
Nucleotide NucleotideNucleotide Nucleotide
Nucleotide
Nucleotide
Nucleotide
Nucleotide Nucleotide Nucleotide Nucleotide
NucleotideNucleotide Nucleotide
Nucleotide
The Rule: Complimentarity• Adenine always base pairs with Thymine (or
Uracil if RNA)
• Cytosine always base pairs with Guanine.
• This is because these bases are complimentary to each other
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DNA Structure
• The DNA helix is “anti-parallel” – Each strand of the helix has a 5’ (5 prime) end and a 3’ (3 prime) end.
53
DNA Structure
Strand 1
(Watson strand)Strand 2
(Crick strand)
5 ‘ end
3 ‘ end
3’ end
5’end
Types of DNA-Three helical forms of DNA are recognized to exist: A, B, and
Z forms.
1-The B conformation is the dominate form under physiological conditions.
-In B DNA, the base pairs are stacked 0.34 nm apart, with 10
base pairs per turn of the right-handed double helix and a diameter of approx 2 nm .
2-Like B DNA, the A DNA is also a right-handed helix. However, A DNA exhibits a larger diameter (2.6 nm), with 11 bases per turn of the helix, and the bases are stacked closer together in the helix (0.25 nm apart) .
3 -In contrast to the A and B forms of DNA, a Z DNA is a left-handed helix. possesses a minor groove but no major groove, and the minor groove is sufficiently deep that it reaches the axis of the DNA helix.
54
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Ångström (Å)= 10−10 m
56
syn and anti conformations
The bond joining the 1′-carbon of the deoxyribose sugar to the heterocyclic base is the N-glycosidic bond. Rotation about this bond gives: syn and anti conformations. Rotation about this bond is restricted and the anti conformation is generally favoured, partly on steric grounds.
Types of DNA57
The Denaturation (Melting) of DNA
-The double-stranded structure of DNA can be separated into two component strands (melted) in solution by increasing the temperature or decreasing the salt concentration.
-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
58
-The strands of a given molecule of DNA separate over a
temperature range. The midpoint is called the melting temperature,
or Tm .
The Tm is influenced by the base composition of the DNA and by
the salt concentration of the solution. DNA rich in G–C pairs, which
have three hydrogen bonds, melts at a higher temperature than that
rich in A–T pairs, which have two hydrogen bonds.
59
FUNCTIONS OF DNA
-Stores genetic information.-Maintains growth and repair.-Controls all cellular activities.-Contains protein codes.-Ensures each daughter cell & gamete receives exact genetic information.
60
THE ORGANIZATION OF DNA IN CHROMOSOMES
61
Structure of chromosome, chromatin and nucleosome
-Chromatin consists of very long double-stranded DNA molecules and a nearly equal mass of rather small basic proteins (Lysine and arginine) termed histones as well as a smaller amount of nonhistone proteins (most of which are acidic and larger than histones) and a small quantity of RNA.
-Electron microscopic studies of chromatin havedemonstrated dense spherical particles called nucleosomes,which are approximately 10 nm in diameter and connected by DNA filaments.
-Nucleosomes are composed of DNA wound around a collection of histone molecules. 62
Chromosome
Supercoils
Coils
Nucleosome
Histones
DNAdoublehelix
63
Chromosome: It is a linear strand of DNA in combination with nuclear proteins. refer to this complex of DNA and proteins as (so called chromatin) It is a linear array of genes as a set – they are our genome.
In human 23 pairs of chromosomes.
What is a chromosome?
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65
Chromosome is a compact form of the DNA that readily fits inside the cell
To protect DNA from damageDNA in a chromosome can be
transmitted efficiently to both daughter cells during cell division
Chromosome confers an overall organization to each molecule of DNA, which facilitates gene expression as well as recombination
**Genes are regions within a chromosome .- Like words within a sentence.
66
The genome is the genetic material of an organism. It consists of DNA (or RNA in RNA viruses). The genome includes both the genes and the non-coding sequences of the DNA/RNA
Genome
Human genome stored on DNA is that of instructions stored in a book:
The book (genome) would contain 23 chapters (chromosomes)
Each chapter contains 48 to 250 million letters (A,C,G,T) without spaces;
Hence, the book contains over 3.2 billion letters total;
The book fits into a cell nucleus the size of a pinpoint;
•At least one copy of the book (all 23 chapters) is contained in most cells of our body. The only exception in humans is found in mature red blood cells which become enucleated during development and therefore lack a genome.
67
Part of DNA sequence of
complete genome of virus
Sequence complexity refers to the number of times a particular base sequence appears in the genome
There are three main types of repetitive sequencesUnique or non-repetitiveModerately repetitiveHighly repetitive
Repetitive Sequences
68
Unique or non-repetitive sequencesFound once or a few times in the genome Includes structural genes as well as intergenic areas
Moderately repetitiveFound a few hundred to a few thousand times Includes
• Genes for rRNA and histones• Origins of replication• Transposable elementsmicrosatellites / minisatellites (DNA 'fingerprints)
Highly repetitive Found tens of thousands to millions of times Each copy is relatively short (a few nucleotides to several hundred in
length) eg. alpha-satellite DNA - used to construct artificial chromosome
Repetitive Sequences
69
RNA
Almost all single stranded (exception is RNAi). *RNAi (RNA interference) In some RNA molecules (tRNA) many of the bases are
modified (e.g. pseudouridine). Has capacity for enzymatic function -ribozymes One school of thought holds that early organisms were
based on RNA instead of DNA (RNA world).
70
RNA TYPES Several different “types” which reflect
different functions mRNA (messenger RNA)
tRNA (transfer RNA)
rRNA (ribosomal RNA)
snRNA (small nuclear RNA)
RNAi (RNA interference)
hnRNA is heterogeneous nuclear RNA 71
RNA functions mRNA – transfers information from DNA to
ribosome (site where proteins are made) tRNA – “decodes” genetic code in mRNA, inserts
correct A.A. in response to genetic code. rRNA-structural component of ribosome snRNA-involved in processing of mRNA RNAi-double stranded RNA, may be component of
antiviral defense mechanism. hnRNA- Precursor of mRNA (pre-mRNA), also
termed heterogeneous nuclear RNA (hnRNA).72
RNA Structure
-Messenger RNAs, particularly in eukaryotes, have
some unique chemical characteristics .
-The 5 terminal of mRNA is “capped” by a 7-
methylguanosine triphosphate-The cap is involved in the recognition of mRNA by
the translating machinery, and it probably helps
stabilize the mRNA by preventing the attack of 5-
exonucleases.
**Some footnote :
73
-The other end of most mRNA molecules, the 3-
hydroxyl
terminal, has an attached polymer of adenylate
residues 20–250 nucleotides in length. The specific
function of the poly(A)
“ tail” at the 3-hydroxyl terminal of mRNAs.-Poly(A) “tail” maintains the intracellular stability
mRNA by preventing the attack of 3-exonucleases
74
-tRNA has anticodon that base pairs with the codon in mRNA and carries an amino acid corresponding to that codon (at amino acid binding site
Codon: 3base sequence codes for specific amino acid.
Through adapter molecules: tRNA
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DNA vs RNA DNA consists of two associated polynucleotide strands that wind together in a helical fashion. It is often described as a double helix
Most RNA is single stranded and does not form a double helix
Differences between DNA and RNA
DNA vs RNADNA CompositionDeoxyribose (a pentose = sugar with 5 carbons)
Phosphoric Acid
Organic (nitrogenous) bases:
(Purines - Adenine and Guanine, or Pyrimidines -Cytosine and Thymine)
RNA CompositionRibose (a pentose = sugar with 5 carbons)
Phosphoric Acid
Organic (nitrogenous) bases:
Purines (Adenine and Guanine) and Pyrimidines (Cytosine and Uracil)
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DNA vs RNADNA CompositionThe base composition is variable, but in all cases the amount of adenine is equal to the amount of thymine (A=T).
In the same manner, C=G.
Consequently
A+C = T+G
RNA CompositionThe rule A+C=U+G CAN'T BE
APPLIED THERE
because most RNA is single stranded and does not form a
double helix
The Central Dogma of molecular Biology
79
Central Dogma
• Replication• DNA making a copy of itself
• Making a replica• Transcription
• DNA being made into RNA• Still in nucleotide language
• Translation• RNA being made into protein
• Change to amino acid language
80
Nucleic Acid to Protein•-How does the information in
codons of mRNA get translated into amino acids
in polypeptides?
•-Through adapter molecules: tRNA
-tRNA has anticodon that base pairs with the codon in mRNA and carries an amino acid corresponding to that codon.
Summary: -Codon in mRNA-Anticodon in tRNA-Codon: 5’- 1-2-3 -3’-Anticodon: 3’- 3-2-1 -5’ 81
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Protein Synthesis
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