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SECTION C Properties of Nucleic Acids Molecular Biology Course.

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Presentation on theme: "SECTION C Properties of Nucleic Acids Molecular Biology Course."— Presentation transcript:

1 SECTION C Properties of Nucleic Acids Molecular Biology Course

2 The Race for the Double Helix (1994)

3 The diffraction pattern from the DNA optical transform slide shows the central cross pattern indicative of the helical arrangement of the strands of DNA. It also shows a missing 4th layer line which is the result of the two strands of the double helix being offset by 3/8 of a period. The strongly diffracting phosphorus atoms create the diamonds. The satelites above and below the cross are attributed to the base pairs.

4 C1 Nucleic Acid Structure (DNA & RNA) : bases  nucleosides  nucleotide  phosphodiester bonds  primary sequence  structure, modified nucleic acids Molecular Biology Course C2 Chemical and Physical Properties of Nucleic Acids (DNA & RNA): stability (force), chemical properties (acid, alkali, chemical denaturation), physical properties (viscosity, buoyant density) C3 Spectroscopic and Thermal Properties of Nucleic Acids (DNA & RNA): UV absorption, hyperchromocity, quantitation and purity C4 DNA Supercoiling (DNA): closed circular molecule, supercoiling & energy, topoisomer & topoisomerase

5 C. Properties of nucleic acids Bicyclic Purines: Thymine (T) is a 5-methyluracil (U) C1 Nucleic Acid Structure (DNA & RNA) : Bases Monocyclic pyrimidine:

6 The bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside Adenosine, guanosine, cytidine, thymidine, uridine Glycosidic (glycoside, glycosylic) bond ( 糖苷键 ) R Ribose or 2’-deoxyribose C. Properties of nucleic acids C1 Nucleic Acid Structure (DNA & RNA) : Nucleosides

7 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. Deoxynucleotides (containing deoxyribose) Ribonucleotides (containing ribose) Phosphate ester bonds C. Properties of nucleic acids C1 Nucleic Acid Structure (DNA & RNA) : Nucleotides

8 BASESNUCLEOSIDESNUCLEOTIDES Adenine (A) AdenosineAdenosine 5’-triphosphate (ATP) DeoxyadenosineDeoxyadenosine 5’-triphosphate (dATP) Guanine (G) GuanosineGuanosine 5’-triphosphate (GTP) DeoxyguanosineDeoxy-guanosine 5’-triphosphate (dGTP) Cytosine (C) CytidineCytidine 5’-triphosphate (CTP) DeoxycytidineDeoxy-cytidine 5’-triphosphate (dCTP) Uracil (U) UridineUridine 5’-triphosphate (UTP) Thymine (T) Thymidine/ Deoxythymidie Thymidine/deoxythymidie 5’-triphosphate (dTTP) C. Properties of nucleic acids

9 Phosphodiester bond Primary sequence: 5’end: not always has attached phosphate groups 3’ end: free hydroxyl (-OH) group C. Properties of nucleic acids C1 Nucleic Acid Structure (DNA & RNA) : Phosphodiester bonds & primary sequence

10 C. Properties of nucleic acids C1 Nucleic Acid Structure : DNA double helix Watson and Crick, 1953 The genetic material of all organisms except for some viruses The foundation of the molecular biology

11 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 back 5’ 3’ 5’ Sugar-phosphate backbones (negatively charged): outside Planner bases (stack one above the other): inside C. Properties of nucleic acids

12 Base pairing A:T G:C C. Properties of nucleic acids back

13 Helical turn: 10 base pairs/turn 34 A o /turn back C. Properties of nucleic acids

14 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. C. Properties of nucleic acids C1 Nucleic Acid Structure : RNA structure

15 Ribozyme RNA Tertiary structure tRNA Secondary structure

16 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 ProteinNucleic Acids Fibrous proteinGlobular protein Helical DNA Globular RNA Structural proteins Enzymes, antibodies, receptors etc Genetic information maintenance Ribozymes Transfer RNA (tRNA) Signal recognition etc. C. Properties of nucleic acids

17 C1 Nucleic Acid Structure : Modified Nucleic Acids Modifications correspond to numbers of specific roles. We will discuss them in some related topics. For example, methylation of A and C to can avoid restriction digestion of endogenous DNA sequence (Topic G3). C. Properties of nucleic acids

18 C2 Chemical and Physical Properties of Nucleic Acids 1.Stability of Nucleic Acids 2.Effect of Acid & applications 3.Effect of alkali & applications 4.Chemical denaturation 5.Viscosity & applications 6.Buorant density & application Chemical properties Physical properties back Molecular Biology Course

19 Stability of Nucleic Acids 1.Hydrogen bonding Does not normally contribute the stability of nucleic acids or protein Contributes to specific structures of these macromolecules. For example,  -helix,  -sheet, DNA double helix, RNA secondary structure 2. Stacking interaction/hydrophobic interaction between aromatic base pairs/bases contribute to the stability of nucleic acids. It is energetically favorable for the hydrophobic bases to exclude waters and stack on top of each other This stacking is maximized in double-stranded DNA C. Properties of nucleic acids Fig

20 Effect of Acid Strong acid + high temperature: completely hydrolyzed to bases, riboses/deoxyrobose, and phosphate 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 C. Properties of nucleic acids

21 Effect of Alkali & Application 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 C. Properties of nucleic acids 1.High pH (> 7-8) has subtle (small) effects on DNA structure 2.High pH changes the tautomeric ( 互变异构 ) state of the bases

22 RNA hydrolyzes at higher pH because of 2’-OH groups in RNA RNA is unstable at higher pH OH free 5’-OH 2’, 3’-cyclic phosphodiester alkali C. Properties of nucleic acids

23 Chemical Denaturation Urea (H 2 NCONH 2 ) ( 尿素) : denaturing PAGE Formamide (HCONH 2 ) (甲酰胺) : Northern blot Disrupting the hydrogen bonding of the bulk water solution Hydrophobic effect (aromatic bases) is reduced Denaturation of strands in double helical structure C. Properties of nucleic acids

24 Viscosity (粘性) 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. C. Properties of nucleic acids

25 Buoyant density (DNA) 1.7 g cm -3, a similar density to 8M CsCl Purifications of DNA: equilibrium density gradient centrifugation back RNA pellets at the bottom Protein floats C. Properties of nucleic acids

26 1.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 ( max = 260 nm) Applications: detection, quantitation, assessment of purity (A 260 /  280 ) 2. Hypochromicity : caused by the fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide Molecular Biology Course C3 Spectroscopic and Thermal Properties of Nucleic Acids

27 3. Quantitation of nucleic acids Extinction coefficients: 1 mg/ml dsDNA has an A 260 of 20 ssDNA and RNA, 25 The values for ssDNA and RNA are approximate (1)The values are the sum of absorbance contributed by the different bases (  : purines > pyrimidines) (2)The absorbance values also depend on the amount of secondary structures due to hypochromicity. 4.Purity of DNA A 260 /  280 : dsDNA--1.8 pure RNA--2.0 protein--0.5 C. Properties of nucleic acids

28 back C. Properties of nucleic acids 5. 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.

29 6. 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. 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) Fig. 2. C. Properties of nucleic acids

30 DNA Supercoiling C. Properties of nucleic acids Closed circular molecule Supercoiling & energy Topoisomer & topoisomerase

31 1.Almost all DNA molecules in cells can be considered as circular, and are on average negatively supercoiled. Counter helical turn C. Properties of nucleic acids

32 2.Negative supercoiled DNA has a higher torsional ( 扭 转的 ) energy than relaxed DNA, which facilitates the unwinding of the helix, such as during transcription initiation or replication 3.Topoisomer: A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands. C. Properties of nucleic acids

33 Topoisomerases exist in cell to regulate the level of supercoiling of DNA molecules. Type I topoisomerase: breaks one strand and change the linking number in steps of ±1. TypeII topoisomerase: breaks both strands and change the linking number in steps of ±2. Gyrase: introduce the negative supercoiling (resolving the positive one and using the energy from ATP hydrolysis. C. Properties of nucleic acids

34 Ethidium bromide (intercalator): locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe. Topoisomerases Type I : break one strand of the DNA, and change the linking number in steps of ±1. Type II : break both strands of the DNA, and change the linking number in steps of ±2. C. Properties of nucleic acids


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