SECTION C Properties of Nucleic Acids

1. Molecular Biology Course SECTION C Properties of Nucleic Acids

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. Molecular Biology Course C1 Nucleic Acid Structure (DNA & RNA): basesnucleosides nucleotide  phosphodiester bonds  primary sequence  structure, modified nucleic acids 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 C1 Nucleic Acid Structure (DNA & RNA): Bases Bicyclic Purines: Monocyclic pyrimidine: Thymine (T) is a 5-methyluracil (U)

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

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

8. C. Properties of nucleic acids

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

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. C. Properties of nucleic acids Essential for replicating DNA and transcribing RNA • Two separate strandsAntiparellel(5’3’ direction) • Complementary (sequence) • Base pairing: hydrogen bonding that holds two strands together 3’ 5’ • Sugar-phosphate backbones (negatively charged): outside • Planner bases (stack one above the other): inside 3’ 5’ back

12. C. Properties of nucleic acids 4 1 3 2 6 7 5 8 1 9 4 2 3 A:T G:C Base pairing back

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

14. C. Properties of nucleic acids C1 Nucleic Acid Structure : RNA structure • Single stranded nucleic acid • Secondary structure are formed some time • Globular tertiary structure are important for many functional RNAs, such as tRNA, rRNA and ribozyme RNA Forces for secondary and tertiary structure: intramolecularhydrogen bonding and base stacking.

15. Ribozyme RNA tRNA Secondary structure Tertiary structure

16. C. Properties of nucleic acids 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

17. C. Properties of nucleic acids 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).

18. Molecular Biology Course C2 Chemical and Physical Properties of Nucleic Acids • Stability of Nucleic Acids • Effect ofAcid & applications • Effect of alkali & applications • Chemical denaturation • Viscosity & applications • Buorant density & application Chemical properties Physical properties back

19. C. Properties of nucleic acids Stabilityof Nucleic Acids • Hydrogen bonding • Does not normally contribute the stability of nucleic acids or protein • Contributes to specific structures of these macromolecules. For example, a-helix, b-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 Fig

20. C. Properties of nucleic acids Effect ofAcid 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

21. C. Properties of nucleic acids Effect of Alkali & Application • High pH (> 7-8) has subtle (small) effects on DNA structure • High pH changes the tautomeric (互变异构)state of the bases enolate form keto form enolate form keto form Base pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturation

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

23. C. Properties of nucleic acids Chemical Denaturation Urea(H2NCONH2) (尿素）: denaturing PAGE Formamide (HCONH2) （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

24. C. Properties of nucleic acids Viscosity（粘性） • Reasonsfor the DNA high viscosity • High axial ratio • Relatively stiff Applications: Long DNA molecules can easily be shortened by shearing force. Remember to avoid shearing problem when isolating very large DNA molecule.

25. C. Properties of nucleic acids 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 back

26. Molecular Biology Course C3 Spectroscopic and Thermal Properties of Nucleic Acids • 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 (lmax = 260 nm) • Applications: detection, quantitation, assessment of purity (A260/A280) 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

27. C. Properties of nucleic acids • 3. 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 (e : purines > pyrimidines) • The absorbance values also depend on the amount of secondary structures due to hypochromicity. • Purity of DNA • A260/A280: • dsDNA--1.8 • pure RNA--2.0 • protein--0.5

28. 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. back

29. C. Properties of nucleic acids 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. 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)

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

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

32. C. Properties of nucleic acids • 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 • Topoisomer: A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands.

33. C. Properties of nucleic acids Topoisomerasesexist 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.

34. C. Properties of nucleic acids 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.