Chapter 2 Nucleic Acid

•Biochemistry Course

Chapter 2 Nucleic Acid Structure and Analyais

Section 1 Chemical Composition of Nucleic Acids Section 2 DNA Structure Section 3 RNA Structure Section 4 Chemical and Physical Properties of Nucleic Acids

Section 1 Chemical Composition of Nucleic Acid

Comparisons of names of bases, nucleosides and nucleotides BASES

NUCLEOSIDES

NUCLEOTIDES

Adenine (A)

Adenosine

Adenosine 5’-triphosphate (ATP)

Deoxyadenosine

Deoxyadenosine 5’-triphosphate (dATP)

Guanosine

Guanosine 5’-triphosphate (GTP)

Deoxy-guanosine

Deoxy-guanosine 5’-triphosphate (dGTP)

Cytidine

Cytidine 5’-triphosphate (CTP)

Deoxy-cytidine

Deoxy-cytidine 5’-triphosphate (dCTP)

Uracil (U)

Uridine

Uridine 5’-triphosphate (UTP)

Thymine (T)

Thymidine/ deoxythymidine

Thymidine/deoxythymidine 5’-triphosphate (dTTP)

Guanine (G) Cytosine (C)

Purine: A & G; Pyrimidine: C & T/U; (deoxy)-ribose,

Nitrogenous bases

Bicyclic purines:

Monocyclic pyrimidine:

Thymine (T) is 5-methyluracil (U)

Nucleosides In nucleic acids, the bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside Glycosidic (glycoside, glycosylic) bond

R

Ribose or 2’-deoxyribose

Adenosine, guanosine, cytidine, thymidine, uridine

Nucleotides A nucleotide is a nucleoside with one or more phosphate groups bound covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the case of 5’-position, up to three phosphates may be attached. Phosphate diester bonds 7 9

Deoxynucleotides (deoxyribose containing)

5 4

6

1 2

5

4 1 2

Ribonucleotides (ribose containing)

5’end: not always has attached phosphate groups DNA/RNA sequence: From 5’ end to 3’ end Example: 5’-UCAGGCUA-3’ = UCAGGCUA

Phosphodiester bonds

3’ end: free hydroxyl (-OH) group

Section 2 DNA Structure 1. Primary structure of DNA: 5’end: free phosphate group

3’ end: free hydroxyl (-OH) group

Phosphodiester bond

2. Secondary structure of DNA •Watson and Crick , 1953 •The genetic material of all organisms except for some viruses

•The foundation of the molecular biology

•Two separate strands Antiparellel (5’→3’ direction) Complementary (sequence) Base pairing: hydrogen bonding that holds two strands together

Essential for replicating DNA and transcribing RNA

• Sugar-phosphate backbones (negatively charged): outside • Planner bases (stack one above the other): inside back

3’

5’

3’

5’

A:T

G:C

Base pairing back

•Helical turn: 10 base pairs/turn

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Section 3 : RNA structure 1. Single stranded nucleic acid 2. Secondary structure are formed some time 3. Globular tertiary structure are important for many functional RNAs, such as tRNA, rRNA and ribozyme RNA Forces for secondary and tertiary structure: intramolecular hydrogen bonding and base stacking.

tRNA

Ribozyme RNA Secondary structure

Tertiary structure

Conformational variability of RNA is important for the much more diverse roles of RNA in the cell, when compared to DNA. Structure and Function correspondence of protein and nucleic acids

Protein Fibrous protein Globular protein Structural proteins

Nucleic Acids Helical DNA Globular RNA

•Enzymes, Genetic •Ribozymes information •Transfer RNA •antibodies, •receptors etc maintenance (tRNA) •Signal recognition etc.

Section 4 Chemical and Physical Properties of Nucleic Acids 1. Effect of Acid & applications 2.Effect of alkali & applications 3.Chemical denaturation 4.Viscosity & applications 5.Buorant density & application 6.Spectroscopic property 7.Thermal denaturation 8.Renaturation 9.Hybridization

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1.Effect of Acid & Application (1)Strong acid + high temperature: completely hydrolyzed to bases, riboses/deoxyrobose, and phosphate (2)pH 3-4 : apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ], can be generated by formic acid (3)Appication:Maxam and Gilbert DNA sequencing

2.Effect of Alkali & Application (1)pH (7-8) has subtle (small) effects on DNA structure (2)High pH changes the tautomeric state of the bases keto form

enolate form

keto form

enolate form

Base pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturation

RNA hydrolyzes at higher pH because of 2’-OH groups in RNA

2’, 3’-cyclic phosphodiester

alkali

RNA is unstable at higher pH

OH

free 5’-OH

3.Chemical Denaturation & Application Urea (H2NCONH2): denaturing PAGE Formamide (HCONH2): Northern blot Disrupting the hydrogen bonding of the bulk water solution Denaturation of strands in double helical structure

4. Viscosity & Application Reasons for the DNA high viscosity (1) High axial ratio (2) Relatively stiff

Applications: Long DNA molecules can easily be shortened by shearing force. Remember to avoid shearing problem when isolating very large DNA molecule.

5.Buoyant density (DNA) 1.7 g cm-3 , a similar density to 8M CsCl Purifications of DNA: equilibrium density gradient centrifugation

Protein floats

RNA pellets at the bottom

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6.Spectroscopic property & Application UV absorption:

• nucleic acids absorb UV light due to the aromatic bases • The wavelength of maximum absorption by both DNA and RNA is 260 nm (λ max = 260 nm) • Applications: detection, quantitation, assessment of purity (A260 /Α 280 )

(1)Quantitation of nucleic acids Extinction coefficients: 1 mg/ml dsDNA has an A260 of 20 ssDNA and RNA, 25 The values for ssDNA and RNA are approximate The values are the sum of absorbance contributed by the different bases (ε : purines > pyrimidines) The absorbance values also depend on the amount of secondary structures due to hypochromicity.

(2)Purity of DNA and RNA A260 /Α

280

:

dsDNA--1.8 pure RNA--2.0 protein--0.5

7.Thermal denaturation/melting: heating leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA.

RNA: the absorbance increases gradually and irregularly DNA: the absorbance increases cooperatively. melting temperature (Tm): the temperature at which 40% increase in absorbance is achieved.

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8.Renaturation: Rapid cooling: only allow the formation of local base paring. Absorbance is slightly decreased Slow cooling: whole complementation of dsDNA. Absorbance decreases greatly and cooperatively. Fig. 2.

Annealing: base paring of short regions of complementarity within or between DNA strands. (example: annealing step in PCR reaction) Hybridization: renaturation of complementary sequences between different nucleic acid molecules. (examples: Northern or Southern hybridization)