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Chemistry 2223B Topic 6 - Nucleic Acids.pdf

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Chemistry 2223B
James Wisner

Chemistry 2223b  Winter 2013–14  Western University Topic 6. Nucleic Acids  This chapter focuses on some of the biological and chemical aspects of nucleic acids, specifically DNA. We will examine its properties, sequencing, and laboratory synthesis.  Background material that is important includes: o Alcohols o Amines o Strengths of acids o Substitution and elimination reactions Nucleic Acids  2 A. Structure and Composition of Nucleic Acids 1. Nucleobases  N atoms 1 and 9, in the pyrimidines and purines respectively, are used to form N-glycosidic linkages.  The structures shown are the predominant tautomers.  When Watson and Crick worked on DNA structure, their work was hindered because the structures found in old textbooks were of the improper tautomers. Examples of tautomeric pairs: NH NH O OH 2 2 N NH N N N N N N N O N OH H N NH 2 H N NH2 H C (keto form) C (enol form) G (keto form) G (enol form) Nucleic Acids  3 2. Nucleosides  Nucleosides are the1bases attac9ed to sugars (ribose in RNA, 2-deoxyribose in DNA) as -N-glycosides (N pyrimidines, N purines). CHO CHO H OH HOH2C O H H HOH2C O H OH OH H OH OH H OH H OH CH2OH OH OH CH2OH OH D-Ribose 2-Deoxy-D-ribose O NH2 NH N N NH2 HOH C N O HOH C N N 2 O 2 O OH OH OH a ribonucleoside a deoxyribonucleoside Nucleic Acids  4 3. Nucleotides NH 2  Nucleotides are composed N of nucleosides and one or O more phosphates. HO P OH C N O 2 O  Phosphates can be OH attached to one or more of the OH groups (2’, 3’, 5’). OH deoxycytidine-5'-monophosphate  The “prime” refers to the numbering of the sugar. O  The pa1of each phosphate NH group is < 5. O O O N N NH 2 HO P O P O P OH C 2 N  The pa2of each phosphate O (if there is a second protoOH OH OH is about 7. OH OH guanosine triphosphate (GTP) Nucleic Acids  5 4. Nucleic Acids  Nucleic acids are phosphate diesters of nucleotides. DNA has two anti-parallel strands in a right-handed helix. A-T and G-C base pairing by H-bonds. CH 3 H O N H OH -- N N O O 5’-end N H O P O A N O HO O N N H N -- N H O -- O O N O O O P O N O P O C N H N O N O H N H O H N -- H O O O N N O O O-- P N H P O O N O O G H O N N N O H H H N N O O--CHH O N O P 3 OH O N H N N O O T N O O HO 3’-end Nucleic Acids  6  This arrangement forms two grooves that are different in size, termed the major groove and the minor groove. Other molecules can recognize the H-bond donors and acceptors in these grooves and selectively bind to them. major CH3 O H H N T H N N N N A O N N minor major H N H O C N N N H  Other binding methods include non-selective N O G H interactions with the negatively charged phosphate N N N ester backbone, and intercalation, where a molecule H inserts itself between base pairs. minor Intercalation: open up helix and insert molecule/drug in between bases Nucleic Acids  7 Netropsin bound to minor groove Nogalamycin intercalated (one strand shown for clarity in the diagram on the right) A protein bound to the major groove Nucleic Acids  8 5. Reactivity of DNA and RNA in Acids and Bases  Both DNA and RNA can be hydrolyzed under the proper acidic conditions, as they possess acid-labile functional groups (phosphate esters and N-glycosidic linkages).  An important difference between DNA and RNA is that the latter is particularly susceptible to strand cleavage under basic conditions. RO Base RO Base O O O O O O O P O H P O O O Base O HO Base O OR OH OR OH  RNA cleavage in base is accelerated by the presence of divalent cations such as 2+ 2+ 2+ Mg , Cu , Zn , etc. For that reason, purified RNA is usually stored in highly buffered solutions that contain a chelating agent such as EDTA. Nucleic Acids  9 B. DNA Sequencing  Molecular biology and genomics, two fields that involve the study of genes and genomes, underwent explosive growth in the last 10-20 years. This growth would not have been possible without a method to sequence DNA rapidly and accurately.  Sequencing methods were developed in the 1970’s by Sanger and Gilbert, but automated instruments did not appear until the late 1980’s. This instrumentation, invented by analytical chemists, made the Human Genome Project possible. o Sanger won two Nobel Prizes in chemistry for his work on two biological problems: peptide (insulin) sequencing in 1958, DNA sequencing in 1980.  One important property of DNA is fundamental to sequencing: At high temperature, DNA “melts” and separates into its two strands. heat (melt) 5'-GCTATCGGATCG-3' 5'-GCTATCGGATCG-3' 3'-CGATAGCCTAGC-5' + cool (anneal) Double-stranded DNA 3'-CGATAGCCTAGC-5' (H-bonded base pairs) Single-stranded DNA  Because the strands are complementary, only one strand needs to be sequenced. Nucleic Acids  10  DNA sequencing relies on the action of the enzyme DNA polymerase, which uses a strand of DNA as a template and synthesizes the complementary strand using the four 2’-deoxyribonucleotide triphosphates (dNTPs: dATP, dTTP, dCTP, dGTP). o The direction of synthesis by the enzyme is 5’  3’ and is performed by linking the 5’ end of a dNTP to the existing 3’-OH end of the growing chain. o Because DNA polymerase requires an existing 3’-OH group, a small fragment known as a primer is required. After this fragment has annealed to the template strand, DNA polymerase can elongate the primer. 5'-GCT-3' 5'-GCT-3' 3'-CGATAGCCTAGC-5' 3'-CGATAGCCTAGC-5' anneal dNTPs elongate DNApol 5'-GCTATCGGATCG-3' 3'-CGATAGCCTAGC-5' Nucleic Acids  11  The reaction is mechanistically similar to other phosphorylation reactions. O O O O HO P O P O P OH C 2 O base GrowingChain O P OH2C O base O O O 5' O O O OH HO P O P O new 3'H GrowingChain-OH O O 5' 3' 2. Sequencing by Chain Termination  In this popular method, four reaction mixtures are prepared. Each reaction mixture contains: o DNA to be sequenced o Primer 2+ o DNA polymerase, buffer, Mg o All four dNTPs o A small amount of one 2’,3’-dideoxyribonucleotide triphosphate (ddNTP). Nucleic Acids  12  Because ddNTPs do not have a 3’-OH group, they act as chain terminators. If a ddNTP is incorporated into the growing chain, elongation stops. O O O O HO P O P O P OH C base GrowingChain O P OH C base 2 O 5' 2 O O O O O GrowingChain-OH No 3'-OH, cannot 5' 3' add next nucleotide  Each reaction mixture contains all dNTPs and one ddNTP, so the termination point must be at the ddNTP.  However, the termination event occurs only if the ddNTP is randomly incorporated into the growing chain and at the correct location (based on the template strand). Over all 4 something? This results in product chainallf assorted length. Nucleic Acids  13 a. Chain-termination method 1: radioactive ddNTPs  This method requires four separate reactions, one for each base (A, C, G, T). All dideoxy compounds are P-labelled on the P attached to the 5’ oxygen, and that phosphorous is the one incorporated into the chain. O O O O HO P O P O P OH C base GrowingChain O P OH C base 2 O 2 O O O O 5' O No 3'-OH, cannot GrowingChain-OH add next nucleotide 5' 3'  Each reaction contains the DNA to be sequenced, forward primer, DNA polymerase, all four dNTPs, plus a small amount of one of ddATP, ddCTP, ddGTP, or ddTTP. o A reaction: all four dNTPs + some ddATP o C reaction: all four dNTPs + some ddCTP o G reaction: all four dNTPs + some ddGTP o T reaction: all four dNTPs + some ddTTP Nucleic Acids  14  Therefore, in the “A” reaction, termination is only possible after an A and occurs only if ddATP is by chance incorporated, resulting in a radioactive chain. In the example below, three new chains are formed, two of which are radioactive. 5'-GCT-3' Template 5'-GCTA*-3' 3'-CGATAGCCTAGC-5' 5'-GCTATCGGA *3' dNTPs 5'-GCTATCGGATCG-3' ddATP*  The same occurs for the “C” reaction. Note that the first C present in the products is never labelled, because it was added into the mixture as a primer. 5'-GCT-3' Template 5'-GCTATC*-3' 3'-CGATAGCCTAGC-5' 5'-GCTATCGGATC*-3' dNTPs 5'-GCTATCGGATCG-3' ddCTP* Nucleic Acids  15  For the “G” reaction, three radiolabelled chains are formed. Template 5'-GCT-3' 5'-GCTATCG*-3' 3'-CGATAGCCTAGC-5' 5'-GCTATCGG*-3' dNTPs 5'-GCTATCGGATCG*-3' ddGTP* 5'-GCTATCGGATCG-3'  Finally, the T reaction… 5'-GCT-3' Template 5'-GCTAT*-3' 3'-CGATAGCCTAGC-5' 5'-GCTATCGGAT*-3' dNTPs 5'-GCTATCGGATCG-3' ddTTP*  Every radioactive chain is of a different length, allowing separation by size using gel electrophoresis. Photographic film is then placed over the gel, and the decay of P exposes the film, forming a band that corresponds to the location of the chain. Nucleic Acids  16 A reaction A C G T 5'-GCTA -3' * wells 5'-GCTATCGGA*-3' C reaction 5'-GCTATC*-3' 5'-GCTATCGGATC*-3' G reaction 5'-GCTATCG *3' 5'-GCTATCGG*-3' 5'-GCTATCGGATCG*-3' T reaction 5'-GCTAT*-3' 5'-GCTATCGGAT*-3'  Since the primer is known, the sequence can be deduced from the bands. This also allows the determination of
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