1

Dr. Gangadhar ChatterjeeMBBS;MD

Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India

Chemistry of NUCLEIC ACIDS

2

Hundreds of thousands of proteins exist inside each one of us to help carry out our daily functions. These proteins are produced locally, assembled piece-by-piece to exact specifications

3

This information, detailing the specific structure of the proteins inside of our bodies, is stored in a set of

molecules called nucleic acids.

4

Nucleic Acids DNA and RNA

DNA - deoxyribonucleic acidRNA - ribonucleic acid

DNA- stores genetic informationRNA - use in protein synthesis for putting genetic information

5

COMPOSITION OF NUCLEIC ACIDS

Nucleic Acids are POLYMERS

Proteins are polypeptides, Carbohydrates are polysaccharides Nucleic acid is polynucleotide- made of NUCLEOTIDES

6

Monomers

• nucleotides, are made up of three parts:(a) Phosphate (phosphoric acid)(b) N-base (Nitrogenous base)(c) Sugar ~ ribose or deoxyribose

7

Nucleotide and Nucleic Acid Nomenclature

8

Different pentose sugars in RNA & DNA

RNA

DNA

Sugar carbonshave primenumbers, todistinguish themfrom atoms inbases

9

10

11

12

5-Fluorouracil 6-Mercaptopurine

Anticancer agents

Azidothymidine Dideoxyinosine

Antiretroviral agents

Nucleoside and base analogs can be used as anti-cancer and anti-virus drugs

13

14

 It is a linear strand of DNA in combination with nuclear proteins

We refer to this complex of DNA and proteins as chromatin It is a linear array of genes

As a set - they are our genome

What is a chromosome?

15

CHROMOSOMEOrganisms differ in their number of chromosomes

64 chromosomes - 32 pairs

38 chromosomes - 19 pairs

6 chromosomes - 3 pairs

46 chromosomes - 23 pairsHeredity is encoded in DNA within the chromosomes

16

During cell division the DNA is duplicated so that each new cell receives a complete copyEach DNA molecule is made up of many GENESGENE is individual segment of DNA that contains the instructions that direct the synthesis of a single polypeptide

What is a GENE?

17

Central Dogma

How does the sequence of a strand of DNA correspond to the amino acid sequence of a protein? This concept is explained by the central dogma of molecular biology that deals with the detailed residue-by-residue transfer of sequential information, and states that:– information cannot be transferred back from protein to

either protein or nucleic acid.

Replication

• In other words, 'once information gets into protein, it can't flow back to nucleic acid.'

The primary structure of DNA is the sequence

5’ end

3’ end

5’

3’

Phosphodiesterlinkage

Traditionally, a DNA sequence is drawn from 5’ to 3’ end.

A shorthand notation for this sequence is ACGTA

20

The Double Helix (1953)

21

WATSON-CRICK MODELCombination of two single strands

The Double Helix

Sugar-phosphate backbone outside, bases inside

1953

22

WATSON-CRICK MODEL

Bases form specific base pairs, held together by hydrogen bonds

Structure compatible with any sequence of bases

23

WATSON-CRICK MODELThe nucleotide bases of the DNA molecule form complementary pairs: adenine always bonds to thymine (and vice versa) and guanine always bonds to

cytosine (and vice versa). This bonding occurs across the molecule, leading to a double-stranded system

24

Base Pairing in DNADNA samples from different cells of the same species have the same proportions of the four heterocyclic basesDNA samples from different species have different proportions of basesHuman DNA contains:30% - Adenine equal amounts 30% - Thymine A = T20% - Guanine equal amounts 20% - Cytosine G = C

The bases occur in pairs!!!ERWIN CHARGOFF

25

The secondary structure of DNA is the double helix

27

Helical sense: right handed

Base pairs: almost perpendicular to the helix axis; 3.4 Å apart

One turn of the helix: 36 Å; ~10.4 base pairs

Minor groove: 12 Å across

Major groove: 22 Å across

Normally hydrated DNA: B-form DNA

By convention DNA sequence is written from 5’ to 3’

A sequence written from 5’ to 3’ direction is called positive or sense strand.

The other strand in the double helix written from 3’ – 5’ is called negative or antisense strand.

The two are complementary to each other.

  

Structural forms of DNAProperty A-DNA B-DNA Z-DNA

Helix Handedness Right Right Left

Base Pairs per turn 11 10.4 12

Rise per base pair along axis

0.23nm 0.34nm 0.38nm

Pitch 2.46nm 3.40nm 4.56nm

Diameter 2.55nm 2.37nm 1.84nm

Conformation of Glycosidic bond

anti anti Alternating anti and syn

Major Groove Present Present Absent

Minor Groove Present Present Deep cleft

In eukaryotic cells,

DNA is folded into chromatin

DNA Tertiary Structure

•DNA DOUBLE HELICAL STRUCTURE COILS ROUND HISTONES.

•DNA BOUND TO HISTONES FORMS

NUCLEOSOMES (10nm FIBRES)

•NUCLEOSOMES CONTAIN 146 NUCLEOTIDES

Nucleosomes

any of the repeating globular subunits of chromatin that consist of a complex

of DNA and histone

Compaction of DNA in a eukaryotic chromosome

Supercoil = coil over coil

34

Several Characteristics of Nucleic Acids 1. UV absorption:

Nucleic acids absorb UV at a wavelength of 260nm. This feature can be utilized for the analysis and quantification of nucleic acids. 2. Denature and renature Denature of DNA means the breakage of H bonds, unwinding and separation of the two strands.

Reversible denaturation and annealing of DNA

DNA denature can be caused by heat or chemicals.

Denaturation caused by heating is called DNA melting.

The increase in UV absorption due to DNA melting is called hyperchromic shift.  

The temperature required for a DNA to denature is it’s meting temperature (Tm).

Tm varies according the GC content in a DNA molecule. DNA with high GC content is more stable.

How to determine Tm?

When UV absorption of a DNA molecule at 260nm is plotted against the temperature, you get a curve called melting profile of a DNA.

OD 260 – unit of UV absorption.

Temperature DNA denature OD260

DNA denature causes it’s UV absorption to increase:

This curve describes the relationship between UV absorption and temperature.

Tm – melting temperature, the point where 50% of the strands are separated.

A higher Tm represent a higher GC content in the molecule.  

Is DNA denature reversible? Yes.

When heated DNA is slowly cooled down, the two complementary strands will reassociate with each other.

At a temperature near Tm, H-bonds will reform and the double helix is reconstructed. This process is called DNA renature or DNA annealing.  Any two nucleotide strands that share sequence homology can anneal to each other at a right temperature.

It refers to the annealing process between two single stranded polynucleotide strands which are from different sources.  For Exp., an RNA molecule can anneal to the single stranded DNA from which it was transcribed from. The result of DNA/ RNA hybridization confirmed the transcription scheme proposed in 1960s. 

DNA hybridization

Application of DNA hybridization in evolutionary genetics:

A powerful molecular biology technique.

DNA from evolutionally related species would share sequence homology and, therefore, can

hybridize to each other.

Electrophoresis of Nucleic Acids Electrophoresis is a technique that allows the separation of DNA or RNA fragments according to their sizes.  

Functions of DNA and summary of structure

DNA consists of four bases—A, G, C, and T—that are held in linear array by phosphodiester bonds through the 3' and 5' positions of adjacent deoxyribose

moieties.

DNA is organized into two strands by the pairing of bases A to T and G to C on complementary strands. These strands form a double helix around a central axis.

The 3 x 109 base pairs of DNA in humans are organized into the haploid complement of 23 chromosomes.

DNA provides a template for its own replication and thus maintenance of the genotype and for the transcription of the roughly 30,000 human genes into a variety

of RNA molecules.

RNA (Ribonucleic acid )

RNA is a polymer of ribonucleotides linked together by 3’-5’ phosphodiester linkage

RNA V/S DNA

Differences between RNA and DNA

S.No. RNA DNA

1) Single stranded mainly except when self complementary sequences are there it forms a double stranded structure (Hair pin structure)

Double stranded (Except for certain viral DNA s which are single stranded)

2) Ribose is the main sugar The sugar moiety is deoxy ribose

3) Pyrimidine components differ. Thymine is never found(Except tRNA)

Thymine is always there but uracil is never found

4) Being single stranded structure- It does not follow Chargaff’s rule

It does follow Chargaff's rule. The total purine content in a double stranded DNA is always equal to pyrimidine content.

Differences between RNA and DNAS.No. RNA DNA

5) RNA can be easily destroyed by alkalies to cyclic diesters of mono nucleotides.

DNA resists alkali action due to the absence of OH group at 2’ position

6) RNA is a relatively a labile molecule, undergoes easy and spontaneous degradation

DNA is a stable molecule. The spontaneous degradation is very 2 slow. The genetic information can be stored for years together without any change.

7) Mainly cytoplasmic, but also present in nucleus (primary transcript and small nuclear RNA)

Mainly found in nucleus, extra nuclear DNA is found in mitochondria, and plasmids etc

8) The base content varies from 100- 5000. The size is variable.

Millions of base pairs are there depending upon the organism

Differences between RNA and DNAS.No. RNA DNA

9) There are various types of RNA – mRNA, r RNA, t RNA, Sn RNA, Si RNA, mi RNA and hn RNA. These

RNAs perform different and specific functions.

DNA is always of one type and performs the function of storage and

transfer of genetic information.

10) No variable physiological forms of RNA are found. The different types of

RNA do not change their forms

There are variable forms of DNA (A to E and Z)

11) RNA is synthesized from DNA, it can not form DNA(except by the action of

reverse transcriptase). It can not duplicate (except in certain viruses

where it is a genomic material )

DNA can form DNA by replication, it can also form RNA by transcription.

12) Many copies of RNA are present per cell

Single copy of DNA is present per cell.

Types of RNAIn all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist-1) Messenger RNA(m RNA)2) Transfer RNA (t RNA)3) Ribosomal RNA (r RNA)

The other are –o small nuclear RNA (SnRNA),o micro RNA(mi RNA) ando small interfering RNA(Si RNA) ando heterogeneous nuclear RNA (hnRNA).

Messenger RNA (m-RNA)

Comprises only 5% of the RNA in the cellMost heterogeneous in size and base sequenceAll members of the class function as messengers carrying the information in a gene to the protein synthesizing machinery

54

Structural Characteristics of m-RNA

The 5’ terminal end is capped by 7- methyl guanosine triphosphate cap.

The cap is involved in the recognition of mRNA by the translating machinery

It stabilizes m RNA by protecting it from 5’ exonuclease

Structural Characteristics of m-RNA contd.

The 3’end of most m-RNAs have a polymer of Adenylate residues( 20-250)

The tail prevents the attack by 3’ exonucleases

Histones and interferons do not contain poly A tails

On both 5’ and 3’ end there are non coding sequences which are not translated (NCS)

The intervening region between non coding sequences present between 5’ and 3’ end is called coding region. This region encodes for the synthesis of a protein.

Structural Characteristics of m-RNA cont..

5’ cap and 3’ tail impart stability to m-RNA by protecting from specific exonucleases.

Structural Characteristics of m-RNA Contd.The m- RNA molecules are formed with the help of DNA template during the process of transcription.

The sequence of nucleotides in m-RNA is complementary to the sequence of nucleotides on template DNA.

The sequence carried on m -RNA is read in the form of codons.

A codon is made up of 3 nucleotides

The m-RNA is formed after processing of heterogeneous nuclear RNA

Heterogeneous nuclear RNA (hnRNA)

In mammalian nuclei , hnRNA is the immediate product of gene transcription

The nuclear product is heterogeneous in size (Variable) and is very large.

Molecular weight may be more than 107, while the molecular weight of m RNA is less than 2x 106

75 % of hnRNA is degraded in the nucleus, only 25% is processed to mature m-RNA

Heterogeneous nuclear RNA (hnRNA)

Mature m –RNA is formed from primary transcript by capping, tailing, splicing and base modification.

Transfer RNA (t- RNA)Transfer RNA are the smallest of three major species of RNA molecules

They have 74-95 nucleotide residues

They are synthesized by the nuclear processing of a precursor molecule

They transfer the amino acids from cytoplasm to the protein synthesizing machinery, hence the name t RNA.

They are easily soluble , hence called “Soluble RNA or s RNA”

They are also called Adapter molecules, since they act as adapters for the translation of the sequence of nucleotides of the m RNA in to specific amino acids

There are at least 20 species of t RNA one corresponding to each of the 20 amino acids required for protein synthesis.

Structural characteristics of t- RNA

1) Primary structure- The nucleotide sequence of all the t RNA molecules allows extensive intrastand complimentarity that generates a secondary structure.

2) Secondary structure- Each single t- RNA shows extensive internal base pairing and acquires a clover leaf like structure. The structure is stabilized by hydrogen bonding between the bases and is a consistent feature.

Structural characteristics of t-RNA

Secondary structure (Clover leaf structure)All t-RNA contain 5 main arms or loops which are as follows-a) Acceptor armb) Anticodon armc) D HU armd) TΨ C arme) Extra arm

Secondary structure of t- RNAa) Acceptor arm The acceptor arm is at 3’ end It has 7 base pairs The end sequence is unpaired Cytosine, Cytosine-

Adenine at the 3’ end The 3’ OH group terminal of Adenine binds with

carboxyl group of amino acids The t RNA bound with amino acid is called

Amino acyl t RNA CCA attachment is done post transcriptionally

Secondary structure of t- RNA

The carboxyl group of amino acid is attached to 3’OH group of Adenine nucleotide of the acceptor arm. The anticodon arm base pairs with the codon present on the m- RNA

Secondary structure of tRNA contd.b) Anticodon arm Lies at the opposite end of acceptor arm

5 base pairs long

Recognizes the triplet codon present in the m RNA

Base sequence of anticodon arm is complementary to the base sequence of m RNA codon.

Due to complimentarity it can bind specifically with m RNA by hydrogen bonds.

Secondary structure of t- RNA contd.

c) DHU arm It has 3-4 base pairs

Serves as the recognition site for the enzyme (amino acyl t RNA synthetase) that adds the amino acid to the acceptor arm.

d) TΨC armThis arm is opposite to DHU arm

Since it contains pseudo uridine that is why it is so named

It is involved in the binding of t RNA to the ribosomes

Secondary structure of t- RNA(contd.)

e) Extra arm or Variable arm About 75 % of t RNA molecules possess a short extra arm

If about 3-5 base pairs are present the t-RNA is said to be belonging to class 1. Majority t -RNA belong to class 1.

The t –RNA belonging to class 2 have long extra arm, 13-21 base pairs in length.

Tertiary structure of t- RNA

The L shaped tertiary structure is formed by further folding of the clover leaf due to hydrogen bonds between T and D arms.

The base paired double helical stems get arranged in to two double helical columns, continuous and perpendicular to one another.

Ribosomal RNA (rRNA)The mammalian ribosome contains two major nucleoprotein subunits—a larger one with a molecular weight of 2.8 x 106 (60S) and a smaller subunit with a molecular weight of 1.4 x 106 (40S).

The 60S subunit contains a 5S ribosomal RNA (rRNA), a 5.8S rRNA, and a 28S rRNA; there are also probably more than 50 specific polypeptides.

The 40S subunit is smaller and contains a single 18S rRNA and approximately 30 distinct polypeptide chains.

All of the ribosomal RNA molecules except the 5S rRNA are processed from a single 45S precursor RNA molecule in the nucleolus .5S rRNA is independently transcribed.

Ribosomal RNA (rRNA)

Ribosomal RNA (rRNA)The functions of the ribosomal RNA molecules in the ribosomal particle are not fully understood, but they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA to ribosomes and its translation

Recent studies suggest that an rRNA component performs the peptidyl transferase activity and thus is an enzyme (a ribozyme).

Small RNAMost of these molecules are complexed with proteins to form ribonucleoproteins and are distributed in the nucleus, in the cytoplasm, or in both.

They range in size from 20 to 300 nucleotides and are present in 100,000–1,000,000 copies per cell.

Small Nuclear RNAs (snRNAs)snRNAs, a subset of the small RNAs, are significantly involved in mRNA processing and gene regulation

Of the several snRNAs, U1, U2, U4, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA

The U7 snRNA is involved in production of the correct 3' ends of histone mRNA—which lacks a poly(A) tail.

Small Nuclear RNAs (snRNAs)

Sn RNA s are involved in the process of splicing (intron removal) of primary transcript to form mature m RNA. The Sn RNA s form complexes with proteins to form Ribonucleoprotein particles called snRNPs

Micro RNAs (miRNAs), and Small Interfering RNAs (siRNAs)

These two classes of RNAs represent a subset of small RNAs; both play important roles in gene regulation.

miRNAs and siRNAs cause inhibition of gene expression by decreasing specific protein production albeit apparently via distinct mechanisms

Micro RNAs (miRNAs)miRNAs are typically 21–25 nucleotides in length and are generated by nucleolytic processing of the products of distinct genes/transcription units

The small processed mature miRNAs typically hybridize, via the formation of imperfect RNA-RNA duplexes within the 3'-untranslated regions of specific target mRNAs, leading via unknown mechanisms to translation arrest.

Micro RNAs (miRNAs)

microRNAs, short non-coding RNAs present in all living organisms, have been shown to regulate the expression of at least half of all human genes.

These single-stranded RNAs exert their regulatory action by binding messenger RNAs and preventing their translation into proteins.  

Small Interfering RNAs (siRNAs) siRNAs are derived by the specific nucleolytic cleavage of larger, double-stranded RNAs to again form small 21–25 nucleotide-long products.

These short siRNAs usually form perfect RNA-RNA hybrids with their distinct targets potentially anywhere within the length of the mRNA where the complementary sequence exists.

Formation of such RNA-RNA duplexes between siRNA and mRNA results in reduced specific protein production because the siRNA-mRNA complexes are degraded by dedicated nucleolytic machinery;

some or all of this mRNA degradation occurs in specific organelles termed P bodies.

Small Interfering RNAs (siRNAs)

Small interfering RNA (siRNA) are 20-25 nucleotide-long double-stranded RNA molecules that have a variety of roles in the cell. They are involved in the RNA

interference (RNAi) pathway, where it interferes with the expression of a specific gene by hybridizing to its corresponding RNA sequence in the target  mRNA. This

then activates the degrading mRNA. Once the target mRNA is degraded, the mRNA cannot be translated into protein. 

Significance of mi RNAs and si RNAs

Both miRNAs and siRNAs represent exciting new potential targets for therapeutic drug development in humans. In addition, siRNAs are frequently used to decrease or "knock-down" specific protein levels in experimental procedures in the laboratory, an extremely useful and powerful alternative to gene-knockout technology.

SummaryRNA exists in several different single-stranded structures, most of which are directly or indirectly involved in protein synthesis or its regulation.

The linear array of nucleotides in RNA consists of A, G, C, and U, and the sugar moiety is ribose.

The major forms of RNA include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNAs (snRNAs; miRNAs).

Certain RNA molecules act as catalysts (ribozymes).

miRNAs and siRNAs represent exciting new potential targets for therapeutic drug development in humans.

83

FURTHER READING

Reach me @ganga.chatterjee@gmail.com

ganga.chatterjee@outlook.comwww.in.Linkedin.pub>gangadhar >chatterjee

84

THANKS