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nucleic acid notes

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University of British Columbia
CHEM 313

Chem 313: Nucleic Acids We have studied two of the three major kinds of biopolymers: polysaccharides and proteins. Now we will look at the third: nucleic acids. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA contains the genes for heredity; it must be replicated for cell division. In most organisms, the genetic information stored in DNA is transcribed into RNA. This information can then be translated into the synthesis of all the proteins needed for cellular structure and function. DNA was first isolated in 1869 from the nuclei of white blood cells. Because this material was in the nucleus and was acidic, it was called nucleic acid. Nucleotides are also involved in a number of other cellular processes such as energy storage (ATP) and cofactor (NAD/NADH) functions. R'O O n O O P O O Bi O O OP O O B1 n O O R' DNA exists largely as a double helix that is composed of an alternating sugar-phosphate backbone. The polymer contains four monomers (A,C,G,T). Each unit is composed of a pentafuranose (deoxyribose), a purine or pyrimidine base, and a phosphate. The bases are hydrogen bonded to each other via Watson-Crick base pairing. Watson-Crick Base-Pairing R = sugar Hydrogen Bonds H N H H H O N O N N R N N N N H R N N H O N O H N N N N R Thymine (T) R H Cytosine (C) Adenine (A) Guanine (G) 1 O 3 H 9 O N 2 N 4 N NH - 5' B 8 O P O O 1N N O O - N 6 5 N H 7 3OH Pyrimidine Purine Uracil Nomenclature: The bases are often called nucleobases. Thymine, cytosine, and uracil are pyrimidines (1, 3-diazabenzenes). Gaunine and adenine are purines (fused pyrimidine and imidazole). Uracil is found in RNA in place of thymine. (Thymine is more stable than Uracil; T likely evolved from U.) A ribose (RNA) or 2'-deoxyribose (DNA) containing a base (C, A, G, T, or for RNA, U) is called a nucleoside. A unit containing sugar, base, and phosphate is called a nucleotide. Several nucleotides linked together are called oligonucleotides (oligos for short). Many nucleotides together are called nucleic acids: ribonucleic acids (RNA) or deoxyribonucleic acids (DNA). The phosphates are linked at the 3' and 5' positions of the sugars. We won’t worry about the numbering system of the nucleobases, but they get numbers 1, 2, 3, etc., and in order to distinguish them from the sugar carbons, the sugar carbons get numbered 1', 2', 3', etc. NH 2 NH2 N N N N N N O N N HO O H O P O O H O OH H OH H Deoxyribonucleoside Deoxyribonucleotide DNA Synthesis. Why study DNA synthesis? (1) Synthetic DNA is frequently used by chemists, biochemists, and molecular biologists. Non-natural variants of DNA are used for a variety of purposes including as catalysts, drugs, and to probe the origin of life. Enzyme mutants are often generated by incorporating synthetic DNA into plasmids. Likewise for mutation to other compounds such as membrane-bound receptors. Gene- therapy usually involves use of synthetic DNA. (2) Looking at the details of DNA synthesis leads us to examine the structure and chemical properties of DNA more closely. Biosynthesis of DNA: DNA replication is a complex process that we will not discuss in detail. The double helix is opened and the complementary nucleotides are brought into position along each single strand. Over 20 enzymes are involved in the process. In the key step, the 3' hydroxyl of the growing oligo reacts with a 5'-triphosphate of an incoming nucleotide. The leaving group is a pyrophosphate. 2 Biosynthesis of DNA O O P O O O O B P O O i O O O Bi O O O O O P P P O O O O O + O O Bn+1 P n O O pyrophosphate O O Bn+1 3'-hydroxyl O n H O O P O O O O O P P P O Bn+2 O O O O O O O B1 O O R' 5'-triphosphate O R' Overview of chemical synthesis of DNA: The synthesis is via solid phase, similar to peptide synthesis. A nucleotide is bound to the resin at its 3' hydroxyl. (The nucleobases are protected if needed.) Another nucleotide, protected at its 5' hydroxyl and activated at its 3' hydroxyl as a phosphoramidite, is then added, and it is coupled (linked) to the resin- bound nucleotide. The newly formed phosphite group is oxidized to a phosphate, and the protecting group at the (new) 5' hydroxyl is removed. Further coupling followed by cleavage and deprotections yields the oligonucleotide. The 3' and 5' ends of the cleaved oligo can be either hydroxyls (R' = H) or phosphate esters. We’ll see that, as with protein synthesis, DNA biosynthesis proceeds in the opposite direction as DNA chemical synthesis. It’s just that chemists don’t always find nature’s way to be easist in lab. 3 HO O B 1 5' PG O B O O 2 R' O Bn+2 Resin 3' O O OR O P phosphoramidite O P N(iP2) O O O Bi PG O O B2 O O P O O OR P O B n O 1 O B H O n O 1 O B2 Oxiati O 1) tn R' Deprtec O O O 2) Resin P OR 1) More cycles O B O 1 2) Cleavage and Deprotection O Resin Issues that we will try to address: How to get the first nucleotide onto the resin, protection of the bases and the hydroxyls, activation and coupling mechanism, oxidation, cleavage, and deprotection. Note that UBC has a rich history in DNA research. Gobind Khorana was at UBC 1952- 1960. He won the Nobel Prize in 1968 for determining that, in DNA, there is a three nucleotide code for each amino acid. He also went on to do seminal work in oligonucleotide synthesis. Michael Smith (d. 2000), who spent most of his career at UBC, won the Nobel Prize in 1993 for his work in site-directed mutagenesis. Before we ge
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