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.
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
H H O
N O N N R
N N H R N N H O
N O H
N N N N
R Thymine (T) R
H Cytosine (C)
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
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.
N N O N N
O H O P O O H
OH H OH H
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 B P 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
3'-hydroxyl O n
O O O O
P P P O Bn+2
O O O O
O O O
O 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.
O B 1 5'
PG O B
O O 2
R' O Bn+2 Resin 3'
O P phosphoramidite
O P N(iP2)
O Bi PG O
P O O OR
n O 1 O B
H O n O 1
O B2 Oxiati
O 1) tn
R' Deprtec O
O O 2) Resin
1) More cycles
2) Cleavage and Deprotection
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