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Nucleic Acids: Chemistry & Structure Andy Howard Introductory Biochemistry 5 October 2010 Biochemistry:Nucleic Acids I 10/05/2010 Nucleic acids: building blocks Having poked our intellectual noses into proteins, carbohydrates, and lipids, we’re now prepared to investigate nucleic acids—first in terms of the chemistry of the bases and nucleotides, and then in terms of their polymers. 10/05/2010 Biochemistry:Nucleic Acids I p. 2 of 44 What we’ll discuss Nucleic acid chemistry Pyrimidines: C, U, T Purines: A, G Nucleosides & nucleotides Oligo- and polynucleotides DNA duplexes and helicity DNA sequencing 10/05/2010 Biochemistry:Nucleic Acids I p. 3 of 44 Chemistry Nobel Prize 2009 Structural studies of the ribosome Venki Ramakrishnan, LMB Cambridge Thomas Steitz, HHMI Yale University Ada Yonath, Weizmann Institute QuickTime™ and a decompressor are needed to see this picture. QuickTime™ and a dec ompressor are needed to see this picture. 10/05/2010 Biochemistry:Nucleic Acids I QuickTime™ and a dec ompressor are needed to see this picture. p. 4 of 44 6 5 Pyrimidines N 4 2 N 3 Single-ring nucleic acid bases pyrimidine 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 10/05/2010 Biochemistry:Nucleic Acids I 1 p. 5 of 44 H N O 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 10/05/2010 Biochemistry:Nucleic Acids I O HN uracil HN O N H thymine p. 6 of 44 O H N O NH O Tautomers Lactam and Lactim forms Getting these right was essential to Watson & Crick’s development of the DNA double helical model HN O uracil - lactam H HN O N O uracil - lactim HN N H thymine - lactam 10/05/2010 Biochemistry:Nucleic Acids I O O N thymine - lactim p. 7 of 44 OH H N O Cytosine NH2 N cytosine This is 2-oxo,4-aminopyrimidine It’s the other pyrimidine base found in DNA & RNA Spontaneous deamination (CU) Again, other tautomers can be drawn 10/05/2010 Biochemistry:Nucleic Acids I p. 8 of 44 Cytosine: amino and imino forms Again, this tautomerization needs to be kept in mind H N O NH2 H N O N cytosine -amino form NH N cytosine -imino form 10/05/2010 Biochemistry:Nucleic Acids I p. 9 of 44 7 6 5 1N Purines 8 4 2 N 3 H N N 9 Derivatives of purine; again, the purine 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 10/05/2010 Biochemistry:Nucleic Acids I p. 10 of 44 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 NH NH2 H N N N N adenine - amino form H N HN N N adenine - imino form 10/05/2010 Biochemistry:Nucleic Acids I p. 11 of 44 Guanine This is 2-amino-6-oxopurine Found in RNA, DNA Lactam, lactim forms OH O H N H N N HN H2N N guanine - lactam N H2N N N guanine - lactim 10/05/2010 Biochemistry:Nucleic Acids I p. 12 of 44 Other natural purines Hypoxanthine and xanthine are biosynthetic precursors of A & G Urate is important in nitrogen excretion pathways 10/05/2010 Biochemistry:Nucleic Acids I p. 13 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 14 of 44 O HO Nucleosides NR1R2 OH HO N-glycoside of ribofuranose 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) 10/05/2010 Biochemistry:Nucleic Acids I p. 15 of 44 Pyrimidine nucleosides Drawn here in amino and lactam forms OH OH HO HO OH O N H2N N OH O O N O N H cytidine 10/05/2010 Biochemistry:Nucleic Acids I O uridine p. 16 of 44 OH Pyrimidine deoxynucleosides OH H H OH O N O N H OH O OH 2'-deoxyuridine O N H OH N O 2'-deoxythymidine O N H2N O N O deoxycytidine 10/05/2010 Biochemistry:Nucleic Acids I p. 17 of 44 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. 10/05/2010 Biochemistry:Nucleic Acids I p. 18 of 44 Purine nucleosides Drawn in amino and lactam forms NH2 O N N N HN N N H2N N N O O HO HO OH OH HO adenosine 10/05/2010 Biochemistry:Nucleic Acids I HO guanosine p. 19 of 44 Purine deoxynucleosides O NH2 N N HN N N H2N N N N O O OH OH HO deoxyadenosine 10/05/2010 Biochemistry:Nucleic Acids I HO deoxyguanosine p. 20 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 21 of 44 Glycosidic bonds This illustrates the roughly perpendicular positionings of the base and sugar rings 10/05/2010 Biochemistry:Nucleic Acids I p. 22 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 23 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 24 of 44 Monophosphorylated nucleosides NH2 N N N We have specialized names for HO the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates: They are nucleotides N O OO P HO O adenylate Adenosine 5’-monophosphate = AMP = adenylate GMP = guanylate CMP = cytidylate UMP = uridylate 10/05/2010 Biochemistry:Nucleic Acids I O- p. 25 of 44 pKa’s for base N’s and PO4’s Nucleotide pKa base-N pK1 of PO4 pK2 of PO4 5’-AMP 3.8(N-1) 0.9 6.1 5’-GMP 9.4 (N-1) 0.7 6.1 2.4 (N-7) 5’-CMP 4.5 (N-3) 0.8 6.3 5’-UMP 9.5 (N-3) 1.0 6.4 10/05/2010 Biochemistry:Nucleic Acids I p. 26 of 44 UV absorbance These aromatic rings absorb around 260 10/05/2010 Biochemistry:Nucleic Acids I p. 27 of 44 Deoxynucleotides O Similar nomenclature dAMP = deoxyadenylate dGMP = deoxyguanylate dCMP = deoxycytidylate dTMP (= TMP) = deoxythymidylate = thymidylate N HN H2N N 10/05/2010 Biochemistry:Nucleic Acids I N O OO OP HO O deoxyguanylate p. 28 of 44 Di and triphosphates Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety O N O H2N O O O P P P O N O O- O O- OH O- Mg2+ OH HO cytidine triphosphate 10/05/2010 Biochemistry:Nucleic Acids I p. 29 of 44 Cyclic phosphodiesters 3’ and 5’ hydroxyls are both involved in -O-P-O bonds cAMP and cGMP are the important ones (see earlier in the course!) 10/05/2010 Biochemistry:Nucleic Acids I p. 30 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 31 of 44 Typical DNA dinucleotide Various notations: this is pdApdCp Leave out the p’s if there’s a lot of them! -O OP O O O -O N O- N P O O O O N -O P O HN O NH2 O N O 10/05/2010 Biochemistry:Nucleic Acids I N NH2 p. 32 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 33 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 34 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 35 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I Courtesy dnareplication.info p. 36 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 37 of 44 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) 10/05/2010 Biochemistry:Nucleic Acids I p. 38 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 39 of 44 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] 10/05/2010 Biochemistry:Nucleic Acids I p. 40 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 41 of 44 Supercoiling and flat DNA Diagram courtesy SIU Carbondale 10/05/2010 Biochemistry:Nucleic Acids I p. 42 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 43 of 44 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 10/05/2010 Biochemistry:Nucleic Acids I p. 44 of 44