Nucleic acid chemistry and metabolism

1. Nucleic acid chemistry and metabolism Andy HowardBiochemistry Lectures, Fall 201022 November 2010 Nucleic Acid chem&hydrolysis

2. Nucleic Acids • We’ll endeavor to recognize the chemical properties of nucleic acid bases, nucleosides, and nucleotides; and the ways that they behave as oligomers and polymers • Then we’ll look at their biosynthesis Nucleic Acid chem&hydrolysis

3. Nucleic acid chemistry Purines: A, G Other purines Nucleosides Nucleotides Oligo- and polynucleotides Duplex DNA & Helicity DNA sequencing DNA secondary structure Folding kinetics RNA: structure & types DNA & RNA Hydrolysis RNA, DNA Restriction enzymes What we’ll cover Nucleic Acid chem&hydrolysis

4. Chemistry Nobel Prize 2009 • Structural studies of the ribosome • Venki Ramakrishnan, LMB Cambridge • Thomas Steitz, HHMI Yale University • Ada Yonath, Weizmann Institute Nucleic Acid chem&hydrolysis

5. 7 6 5 1 8 4 Purines 2 9 3 • Derivatives of purine; again, the root molecule isn’t biologically important • Six-membered ring looks a lot like pyrimidine • Numbering works somewhat differently: note that the glycosidic bonds will be to N9, whereas it’s to N1 in pyrimidines Nucleic Acid chem&hydrolysis

6. Adenine • This is 6-aminopurine • Found in RNA and DNA • We’ve seen how important adenosine and its derivatives are in metabolism • Tautomerization happens here too Nucleic Acid chem&hydrolysis

7. Guanine • This is 2-amino-6-oxopurine • Found in RNA, DNA • Lactam, lactim forms Nucleic Acid chem&hydrolysis

8. Other natural purines • Hypoxanthine and xanthine are biosynthetic precursors of A & G • Urate is important in nitrogen excretion pathways Nucleic Acid chem&hydrolysis

9. Tautomerization and H-bonds • Lactam forms predominate at neutral pH • This influences which bases are H-bond donors or acceptors • Amino groups in C, A, G make H-bonds • So do ring nitrogens at 3 in pyrimidines and 1 in purines • … and oxygens at 4 in U,T, 2 in C, 6 in G Nucleic Acid chem&hydrolysis

10. Nucleosides • As mentioned in ch. 8, these are glycosides of the nucleic acid bases • Sugar is always ribose or deoxyribose • Connected nitrogen is: • N1 for pyrimidines (on 6-membered ring) • N9 for purines (on 5-membered ring) Nucleic Acid chem&hydrolysis

11. Pyrimidine nucleosides • Drawn here in amino and lactam forms Nucleic Acid chem&hydrolysis

12. Pyrimidine deoxynucleosides Nucleic Acid chem&hydrolysis

13. A tricky nomenclature issue • Remember that thymidine and its phosphorylated derivatives ordinarily occur associated with deoxyribose, not ribose • Therefore many people leave off the deoxy- prefix in names of thymidine and its derivatives: it’s usually assumed. • Exception: T’s in tRNA TC arm Nucleic Acid chem&hydrolysis

14. Purine nucleosides • Drawn in amino and lactam forms Nucleic Acid chem&hydrolysis

15. Purine deoxynucleosides Nucleic Acid chem&hydrolysis

16. Conformations around the glycosidic bond • Rotation of the base around the glycosidic bond is sterically hindered • In the syn conformation there would be some interference between the base and the 2’-hydroxyl of the sugar • Therefore pyrimidines are always anti, and purines are usually anti • Furanose and base rings are roughly perpendicular Nucleic Acid chem&hydrolysis

17. Glycosidic bonds • This illustrates the roughly perpendicular positionings of the base and sugar rings Nucleic Acid chem&hydrolysis

18. Solubility of nucleosides and lability of glycosidic linkages • The sugar makes nucleosides more soluble than the free bases • Nucleosides are generally stable to basic hydrolysis at the glycosidic bond • Acid hydrolysis: • Purines: glycosidic bond fairly readily hydrolyzed • Pyrimidines: resistant to acid hydrolysis Nucleic Acid chem&hydrolysis

19. Chirality in nucleic acids • Bases themselves are achiral • 3 asymmetric centers in ribose • 2 in deoxyribose • Glycosidic bond gives us 1 more,so there are 4 for ribonucleosides,3 for deoxyribonucleosides • Same for nucleotides:phosphates don’t add asymmetries Nucleic Acid chem&hydrolysis

20. Mono-phosphorylated nucleosides • We have specialized names for the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates: • They are nucleotides • Adenosine 5’-monophosphate = AMP = adenylate • GMP = guanylate • CMP = cytidylate • UMP = uridylate Nucleic Acid chem&hydrolysis

21. pKa’s for base N’s and PO4’s Nucleic Acid chem&hydrolysis

22. UV absorbance • These aromatic rings absorb around 260 Nucleic Acid chem&hydrolysis

23. Deoxynucleotides • Similar nomenclature • dAMP = deoxyadenylate • dGMP = deoxyguanylate • dCMP = deoxycytidylate • dTTP (= TTP) = deoxythymidylate = thymidylate Nucleic Acid chem&hydrolysis

24. Di and triphosphates • Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety Nucleic Acid chem&hydrolysis

25. Cyclic phospho-diesters • 3’ and 5’ hydroxyls are both involvedin -O-P-O bonds • cAMP and cGMP are the important ones(see earlier in the course!) Nucleic Acid chem&hydrolysis

26. Oligomers and Polymers • Monomers are nucleotides or deoxynucleotides • Linkages are phosphodiester linkages between 3’ of one ribose and 5’ of the next ribose • It’s logical to start from the 5’ end for synthetic reasons Nucleic Acid chem&hydrolysis

27. Typical DNA dinucleotide • Various notations: this is pdApdCp • Leave out the p’s if there’s a lot of them! Nucleic Acid chem&hydrolysis

28. DNA structure • Many years of careful experimental work enabled fabrication of double-helical model of double-stranded DNA • Explained [A]=[T], [C]=[G] • Specific H-bonds stabilize double-helical structure: see fig. 19.12 Nucleic Acid chem&hydrolysis

29. What does double-stranded DNA really look like? • Picture on previous slide emphasizes only the H-bond interactions • Fig.19.12 is better: shows the tilt of the sugars • Planes of the bases are almost perpendicular to the helical axes on both sides of the double helix Nucleic Acid chem&hydrolysis

30. Sizes (see fig. 19.14) • Diameter of the double helix: 2.37nm • Length along one full turn:10.4 base pairs = pitch = 3.40nm • Distance between stacked base pairs = rise = 0.33 nm • Major groove is wider and shallower;minor groove is narrower and deeper Nucleic Acid chem&hydrolysis

31. What stabilizes this? • Variety of stabilizing interactions • Stacking of base pairs • Hydrogen bonding between base pairs • Hydrophobic effects (burying bases, which are less polar) • Charge-charge interactions:phosphates with Mg2+ and cationic proteins Courtesy dnareplication.info Nucleic Acid chem&hydrolysis

32. How close to instability is it? • Pretty close. • Heating DNA makes it melt: fig. 19.17 • The more GC pairs, the harder it is to melt • Weaker stacking interactions in A-T • One more H-bond per GC than per AT Nucleic Acid chem&hydrolysis

33. iClicker quiz • 1. What positions of a pair of aromatic rings leads to stabilizing interactions? • (a) Parallel to one another • (b) Perpendicular to one another • (c) At a 45º angle to one another • (d) Both (a) and (b) • (e) All three: (a), (b), and ( c) Nucleic Acid chem&hydrolysis

34. 2nd iClicker question! 2. Which has the highest molecular mass among the compounds listed here? • (a) cytidylate • (b) thymidylate • (c) adenylate • (d) adenosine triphosphate • (e) they’re all the same MW Nucleic Acid chem&hydrolysis

35. iClicker quiz, question 3 What would be a suitable wavelength for spectrophotometric detection of nucleic acids? • (a) 230nm • (b) 260 nm • (c) 280 nm • (d) 340 nm • (e) none of the above. Nucleic Acid chem&hydrolysis

36. iClicker quiz, question 4 Which of these RNA octamers would be the most susceptible to acid hydrolysis of the glycosidic linkages? • (a) AUCGAUGU • (b) CUAUCCUC • (c) GCUAGAUG • (d) CGAUGCUA • (e) None of these are susceptible to acid hydrolysis. Nucleic Acid chem&hydrolysis

37. Base composition for DNA • As noted, [A]=[T], [C]=[G] because of base pairing • [A]/[C] etc. not governed by base pairing • Can vary considerably (table 19.2) • E.coli : [A], [C] about equal • Mycobacterium tuberculosis: [C] > 2*[A] • Mammals: [C] < 0.74*[A] • These rules don’t apply to RNA at all, since it isn’t base-paired Nucleic Acid chem&hydrolysis

38. Supercoiling • Refers to levels of organization of DNA beyond the immediate double-helix • We describe circular DNA as relaxed if the closed double helix could lie flat • It’s underwound or overwound if the ends are broken, twisted, and rejoined. • Supercoils restore 10.4 bp/turn relation upon rejoining: see fig. 19.19. Nucleic Acid chem&hydrolysis

39. Supercoiling and flat DNA Diagram courtesy SIU Carbondale Nucleic Acid chem&hydrolysis

40. Sanger dideoxy method • Incorporates DNA replication as an analytical tool for determining sequence • Uses short primer that attaches to the 3’ end of the ssDNA, after which a specially engineered DNA polymerase operates on the DNA • Each vial includes one dideoxyXTP and 3 ordinary dXTPs; the dideoxyXTP will be incorporated but will halt synthesis because the 3’ position is blocked. • See box 20.1 for details Nucleic Acid chem&hydrolysis

41. Automating dideoxy sequencing • Laser fluorescence detection allows for primer identification in real time • An automated sequencing machine can handle 4500 bases/hour • That’s one of the technologies that has made large-scale sequencing projects like the human genome project possible Nucleic Acid chem&hydrolysis

42. DNA secondary structures • If double-stranded DNA were simply a straight-legged ladder: • Base pairs would be 0.6 nm apart • Watson-Crick base-pairs have very uniform dimensions because the H-bonds are fixed lengths • But water could get to the apolar bases • So, in fact, the ladder gets twisted into a helix. • The most common helix is B-DNA, but there are others. B-DNA’s properties include: • Sugar-sugar distance is still 0.6 nm • Helix repeats itself every 3.4 nm, i.e. 10 bp Nucleic Acid chem&hydrolysis

43. Properties of B-DNA • Spacing between base-pairs along helix axis = 0.34 nm • 10 base-pairs per full turn • So: 3.4 nm per full turn is pitch length • Major and minor grooves, as discussed earlier • Base-pair plane is almost perpendicular to helix axis Nucleic Acid chem&hydrolysis

44. Major groove in B-DNA • H-bond between adenine NH2 and thymine ring C=O • H-bond between cytosine amine and guanine ring C=O • Wide, not very deep Nucleic Acid chem&hydrolysis

45. Minor groove in B-DNA • H-bond between adenine ring N and thymine ring NH • H-bond between guanine amine and cytosine ring C=O • Narrow but deep Nucleic Acid chem&hydrolysis

46. Cartoon of AT pair in B-DNA Nucleic Acid chem&hydrolysis

47. Cartoon of CG pair in B-DNA Nucleic Acid chem&hydrolysis

48. What holds duplex B-DNA together? • H-bonds (but just barely) • Electrostatics: Mg2+ –PO4-2 • van der Waals interactions • - interactions in bases • Solvent exclusion • Recognize role of grooves in defining DNA-protein interactions Nucleic Acid chem&hydrolysis

49. Helical twist • Rotation about the backbone axis • Successive base-pairs rotated with respect to each other by ~ 32º Nucleic Acid chem&hydrolysis

50. Propeller twist • Improves overlap of hydrophobic surfaces • Makes it harder for water to contact the less hydrophilic parts of the molecule Nucleic Acid chem&hydrolysis