Nucleic acid chemistry

University of BenghaziFaculty of medicine

Department of Biochemistry

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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

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Two types of nucleic acid are found

• Deoxyribonucleic acid (DNA)• Ribonucleic acid (RNA)

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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

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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

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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”

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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

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There are also derivative of purine bases are:

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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

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• 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

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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

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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

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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'

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. 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 .

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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..

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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..

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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

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Synthetic purine and pyrimidine analogs

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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

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The Double Helix (1953)

© Dr Kalju Kahn USBC Chemistry and BiochemistryPublic Domain image

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Schematic representation of the nucleotide sequences of nucleic acids

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Complementarity of strands in the DNA double helix

Watson-Crick model for the structure of DNA

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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)

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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:

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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.

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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.

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Ångström (Å)= 10−10 m

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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

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-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.

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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.

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THE ORGANIZATION OF DNA IN CHROMOSOMES

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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

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 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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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.

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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.

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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

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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

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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).

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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 :

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-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

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-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

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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

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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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