Nitrogenous bases

1. Nitrogenous bases Bicyclic purines Monocyclic pyrimidine Thymine (T) is 5-methyluracil (U)

2. 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 (糖苷键) R Ribose or 2’-deoxyribose

3. C1Nucleic AcidStructure 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 4 6 7 5 5 1 2 2 9 4 1 Deoxynucleotides (deoxyribose containing) Ribonucleotides (ribose containing)

4. C1Nucleic AcidStructure 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

5. C1Nucleic AcidStructure DNA double helix • Watson and Crick , 1953. • 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 • Planar bases (stack one above the other): inside back

6. 5 5 4 4 6 6 3 3 1 1 2 7 2 7 6 6 5 8 5 1 8 1 9 4 4 9 2 2 3 3 A:T G:C Base pairing via hydrogen bonds

7. C1Nucleic AcidStructure • Double helix • B form: • Right-handed • 10 base pairs/turn • 34 Å/turn • Diameter: ca. 20 Å • Other forms: • A: 11 bases/turn, base plate 20° slant • Z: 12 bases/turn, left-handed helical, one groove

8. C1Nucleic AcidStructure RNA Secondary Structure Single stranded, no long helical structure like double-stranded DNA Globular conformationwith local regions of helical structure formed byintramolecular hydrogen bonding and base stacking. tRNA (clover-like)

9. C1Nucleic AcidStructure Conformational variabilityof RNA is important for the much morediverse rolesof RNA in the cell, when compared to DNA. Structure and Function correspondence of protein and nucleic acids

10. C1Nucleic AcidStructure 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 avoid restriction digestion of endogenous DNA sequence (Topic G3).

11. C2 Chemical and Physical Properties of Nucleic Acids Stability of Nucleic Acids • Hydrogen bonding • Contribute to specificity, not overall stability of DNA helix • Stability lies in the stacking interactions between base pairs • 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 (base stacking & hydrophobic effect). • This stacking is maximized in double-stranded DNA

12. C2Chemical and PhysicalProperties of Nucleic Acids Effect of Acid& applications Strong acid+ high temperature  completely hydrolyzed to (perchloric acid+100°C)bases, riboses/deoxyribose, and phosphate pH 3-4 apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ] Maxam and Gilbert chemical DNA sequencing: A DNA sequencing technique based on chemical removal and modification of bases specifically and then cleaving the sugar-phosphate backbone of the DNA and RNA at particular bases (J2)

13. C2 Chemical and PhysicalProperties of Nucleic Acids Effect ofAlkali & Application DNA denaturationat high pH keto form enolate form keto form enolate form Base pairing is not stable anymore because of the change of tautomeric (异构)statesof the bases, resulting in DNA denaturation

14. Effect of Alkali & Application RNA hydrolyzes 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

15. Chemical Denaturation Urea(H2NCONH2) (尿素）: denaturing PAGE Formamide(HCONH2)（甲酰胺）and Formaldehyde(甲醛): Northern blot Disrupting the hydrogen bonding of the bulk water solution Hydrophobic effect (aromatic bases) is reduced Denaturation of strands in double helical structure

16. Viscosity • Reasons for the DNA high viscosity • High axial ratio • Relatively stiff • Applications • Long DNA molecules can easily be shortened by shearing force. • When isolating very large DNA molecule, always avoid shearing problem

17. Buoyant density 1.7 g cm-3, a similar density to 8M CsCl. Rho=1.66+0.098 (GC)% Purifications of DNA: equilibrium density gradient centrifugation Protein floats RNA pellets at the bottom

18. 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 Fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide

19. 3. Quantitation of nucleic acids Extinction coefficient (e): 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. • Purityof DNA • A260/A280: • dsDNA--1.8 • pure RNA--2.0 • protein--0.5

20. 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.

21. 6. Renaturation: Rapid coolingOnly allow the formation of local base paring Absorbance is slightly decreased Slow cooling Whole complementation of dsDNA. Absorbance decreases greatly and cooperatively. AnnealingBase 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)

22. DNA Supercoiling • Almost all DNA molecules in cells are on average negatively supercoiled. • Supercoiled DNA has a higher energy than relaxed DNA. Negative supercoiling may thus facilitate cellular processes which require the unwinding of the helix, such as transcription initiation or replication • Topoisomerases exist in cell the regulate the level of supercoiling of DNA molecules. (important to know in the sense of gene expression)

23. Linker numbera topological property of a closed-circular DNA, which can be changed only if one or both of the DNA backbones are broken. • Topoisomer A molecule of a given linking number is known as a topoisomer. Topoisomers of the same molecule differ from each other only in their linker number. • The conformation (geometry) of the DNA can be altered while the linking number remains constant. Writhe(wrap around,缠绕)and Twist (扭转) changes are to measure the conformational change of a DNA molecule (Lk = Tw + Wr). • The topological change (Lk) in supercoiling of a DNA molecule is partitioned into a conformational change of twist (Tw )and/or a change of writhe (Wr). • For a given isomer of a circular closed DNA (Lk = 0), the increase in twist will cause a corresponding decrease in writhe.

24. Ethidium bromide(intercalator 插入物) locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe. Topoisomerases Type I break one strand of the DNA (via P-tyrosine bond) , 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. (ATP) Bacterial gyrase (旋转酶) introduce negative supercoiling. ATP.

25. Summary: • Nucleic acid structure • bases > nucleosides (base+sugar) > nucleotides (nucleoside+phosphate) > polynucleotides /DNA/RNA (via 3’,5’-phosphodiester bond) > DNA double helix/RNA secondary structure • Chemical and physical properties • stability support, effect of acid and alkali, chemical denaturation, viscosity, buoyant density • 3. Spectroscopic and thermal properties • 4. DNA supercoiling: Linking number (twist and writhe))