Nucleic Acids: Chemistry & Structure

1. Nucleic Acids:Chemistry & Structure Andy HowardIntroductory Biochemistry8 October 2009 Biochemistry:Nucleic Acids I

2. Nucleic acid chemistry Pyrimidines: C, U, T Purines: A, G Nucleosides & nucleotides Oligo- and polynucleotides DNA duplexes and helicity DNA sequencing DNA secondary structure: A, B, Z Folding kinetics What we’ll discuss Biochemistry:Nucleic Acids I

3. Chemistry Nobel Prize 2009 • Structural studies of the ribosome • Venki Ramakrishnan, LMB Cambridge • Thomas Steitz, HHMI Yale University • Ada Yonath, Weizmann Institute Biochemistry:Nucleic Acids I

4. 6 1 5 Pyrimidines 4 2 3 • Single-ring nucleic acid bases • 6-atom ring; always two nitrogens in the ring, meta to one another • Based on pyrimidine, although pyrimidine itself is not a biologically important molecule • Variations depend on oxygens and nitrogens attached to ring carbons • Tautomerization possible • Note line of symmetry in pyrimidine structure Biochemistry:Nucleic Acids I

5. Uracil and thymine • Uracil is a simple dioxo derivative of pyrimidine: 2,4-dioxopyrimidine • Thymine is 5-methyluracil • Uracil is found in RNA; Thymine is found in DNA • We can draw other tautomers where we move the protons to the oxygens Biochemistry:Nucleic Acids I

6. Tautomers • Lactam and Lactim forms • Getting these right was essential to Watson & Crick’s development of the DNA double helical model Biochemistry:Nucleic Acids I

7. Cytosine • This is 2-oxo,4-aminopyrimidine • It’s the other pyrimidine base found in DNA & RNA • Spontaneous deamination (CU) • Again, other tautomers can be drawn Biochemistry:Nucleic Acids I

8. Cytosine:amino and imino forms • Again, this tautomerization needs to be kept in mind Biochemistry:Nucleic Acids I

9. 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 Biochemistry:Nucleic Acids I

10. 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 Biochemistry:Nucleic Acids I

11. Guanine • This is 2-amino-6-oxopurine • Found in RNA, DNA • Lactam, lactim forms Biochemistry:Nucleic Acids I

12. Other natural purines • Hypoxanthine and xanthine are biosynthetic precursors of A & G • Urate is important in nitrogen excretion pathways Biochemistry:Nucleic Acids I

13. 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 Biochemistry:Nucleic Acids I

14. 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) Biochemistry:Nucleic Acids I

15. Pyrimidine nucleosides • Drawn here in amino and lactam forms Biochemistry:Nucleic Acids I

16. Pyrimidine deoxynucleosides Biochemistry:Nucleic Acids I

17. 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. Biochemistry:Nucleic Acids I

18. Purine nucleosides • Drawn in amino and lactam forms Biochemistry:Nucleic Acids I

19. Purine deoxynucleosides Biochemistry:Nucleic Acids I

20. 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 Biochemistry:Nucleic Acids I

21. Glycosidic bonds • This illustrates the roughly perpendicular positionings of the base and sugar rings Biochemistry:Nucleic Acids I

22. 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 Biochemistry:Nucleic Acids I

23. Chirality in nucleic acids • Bases themselves are achiral • Four asymmetric centers in ribofuranose, counting the glycosidic bond. • Three in deoxyribofuranose • Glycosidic bond is one of those 4 or 3. • Same for nucleotides:phosphates don’t add asymmetries Biochemistry:Nucleic Acids I

24. 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 Biochemistry:Nucleic Acids I

25. pKa’s for base N’s and PO4’s Biochemistry:Nucleic Acids I

26. UV absorbance • These aromatic rings absorb around 260 Biochemistry:Nucleic Acids I

27. Deoxynucleotides • Similar nomenclature • dAMP = deoxyadenylate • dGMP = deoxyguanylate • dCMP = deoxycytidylate • dTTP (= TTP) = deoxythymidylate = thymidylate Biochemistry:Nucleic Acids I

28. Di and triphosphates • Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety Biochemistry:Nucleic Acids I

29. 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!) Biochemistry:Nucleic Acids I

30. 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 Biochemistry:Nucleic Acids I

31. Typical DNA dinucleotide • Various notations: this is pdApdCp • Leave out the p’s if there’s a lot of them! Biochemistry:Nucleic Acids I

32. 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. 10.20 Biochemistry:Nucleic Acids I

33. What does double-stranded DNA really look like? • Picture on previous slide emphasizes only the H-bond interactions; it ignores the orientation of the sugars, which are actually tilted relative to the helix axis • Planes of the bases are almost perpendicular to the helical axes on both sides of the double helix Biochemistry:Nucleic Acids I

34. Sizes (cf fig. 10.20, 11.7) • 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 Biochemistry:Nucleic Acids I

35. 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 Biochemistry:Nucleic Acids I

36. How close to instability is it? • Pretty close. • Heating DNA makes it melt: fig. 11.14 • pH > 10 separates strands too • The more GC pairs, the harder it is to melt DNA thermally • Weaker stacking interactions in A-T • One more H-bond per GC than per AT Biochemistry:Nucleic Acids I

37. iClicker quiz, 1st question • 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) Biochemistry:Nucleic Acids I

38. iClicker question 2 • 2. Which has the highest molecular mass among the compounds listed? • (a) cytidylate • (b) thymidylate • (c) adenylate • (d) adenosine triphosphate • (e) they’re all the same MW Biochemistry:Nucleic Acids I

39. 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 10.3) • E.coli : [A], [C] about equal • Mycobacterium tuberculosis: [C] > 2*[A] • Mammals: [C] < 0.74*[A] Biochemistry:Nucleic Acids I

40. 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 Biochemistry:Nucleic Acids I

41. Supercoiling and flat DNA Diagram courtesy SIU Carbondale Biochemistry:Nucleic Acids I

42. 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 • 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 figs. 11.3 & 11.4 for how these are read out Biochemistry:Nucleic Acids I

43. 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 Biochemistry:Nucleic Acids I

44. 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 Biochemistry:Nucleic Acids I

45. 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 Biochemistry:Nucleic Acids I

46. 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 Biochemistry:Nucleic Acids I

47. 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 Biochemistry:Nucleic Acids I

48. Cartoon of AT pair in B-DNA Biochemistry:Nucleic Acids I

49. Cartoon of CG pair in B-DNA Biochemistry:Nucleic Acids I

50. 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 Biochemistry:Nucleic Acids I