Nucleic Acids
adenosine triphosphate (ATP) a mononucleotide
Nucleic acids consist of: • a ribose (aldopentose) sugar; • a heteroaromatic, glycosidic base;
• a phosphoester. ribonucleic acid (RNA) a polynucleotide
The Ribose Sugar
1
2 5’ cyclizes 3 4’ 1’ Numbering of rings: to 1-n for base; 4 3’ 2’ 1’-5’ for sugar. 5 -D-ribofuranose
D-ribose (an aldopentose) 5 3 1 glycoside cytidine formation cytosine (a nucleoside) (a base) Phosphates and Phosphoesters of Ribonucleosides
a phosphodiester
adenosine (a nucleoside)
adenosine triphosphate a phosphoanhydride (ATP, a nucleotide)
Bases Found in Nucleic Acids
cytosine uracil thymine guanine adenine
cytidine (C) uridine (U) guanosine (G) adenosine (A) Polynucleotides: RNA and DNA
DNA uses thymidine (T), RNA uses uracil (U)
RNA has OH groups at 2’-carbon, DNA does not.
ribonucleic acid (RNA) deoxyribonucleic acid (DNA)
Polynucleotides
• Nucleotide units in DNA and T RNA are linked by 5’ and 3’ oxygens. 5’ • Backbone is negatively charged. 3’ G • Information is stored in sequence of bases. 5’
So, this DNA sequence would read: 3’ C TGC 5’
3’
(typically read 5’ → 3’) RNA Is Less Stable Than DNA
strand break!
2’-OH can serve as DNA lacks a 2’-OH, so it does not intramolecular self-cleave in this way. So,
nucleophile to t½(RNA) = minutes – hours cleave RNA strand t½(DNA) = centuries
Nucleotide Bases Pair with One Another Via Hydrogen Bonding
T A 5’
5’ 3’ 3’ 3’ 5’
5’
3’ 3’ 3’ 5’
G C 5’
Each pair matches one purine with one pyrimidine. Optimal organization for DNA strands is “antiparallel”. Every DNA Sequence Has A Unique, “Complementary” Sequence
5’ 3’
T G C T A
A C G A T
3’ 5’
So, the DNA sequence complementary to 5’-TGCTA-3’ would be 5’-TAGCA-3’
Strands of DNA Form a Right-Handed Double Helix
• DNA base pairs lie flat, stretch across center of helix. • Each turn of the helix is 10 base pairs, 3.4 nm long. 3.4 nm
3.0 nm In Nature, DNA and RNA Are Synthesized by Copying a Single-Strand Template
Addition catalyzed by a DNA/RNA polymerase.
5’ 3’ C Reaction fidelity: > 99% T G
A C G A
3’ 5’
Synthesizing DNA in the Laboratory: Limits of Solution-Phase Synthesis
A typical A • After each synthetic step, solution-phase products must be purified synthesis 1. Add reactants away from reagents, scheme: 2. Purify product unreacted starting material, side products A C • Weakness: Material often 1. Add reactants 2. Purify product lost in purification steps. For long synthesis, these losses multiply. A C G • So, DNA cannot be 1. Add reactants 2. Purify product synthesized chemically by solution-phase methods. A C G T Synthesizing DNA in the Laboratory: Solid-Phase Synthesis
Glass cleavable A • Advantage: Eliminates Bead linker most purification steps. Add reactants, wash • Weakness: Reactions must Glass be extremely high-yield, cleavable A C Bead linker because losses multiply. Add reactants, wash Glass A C G T cleavable A C G Bead linker Add reactants, wash 1. Cleave linker Glass 2. Purify product cleavable A C G T Bead linker
Solid-Phase Synthesis of DNA with Phosphoramidites
dimethoxytrityl (DMT) protecting group
acyl protecting ≡ group cyanoethyl (CNEt) (iPr)2 protecting group diisopropylamine leaving group a phosphoramidite Solid-Phase Synthesis of DNA with Phosphoramidites
(iPr)2 Glass Glass OH Bead Bead tetrazole I2, H2O (w/ linker) (oxidize P) equals
strong H+ (remove DMT) Glass Glass Bead Bead
Solid-Phase Synthesis of DNA with Phosphoramidites
(iPr)2 Glass If yield from oneGlass cycle of OH Bead Bead tetrazolesynthesis is 99% (0.99), I2, H2O (w/ linker) then the yield of a 50- (oxidize P) base DNA molecule is equals (0.99)50 = 61%.
strong H+ (remove DMT) Glass Glass Bead Bead Solid-Phase Synthesis of DNA with Phosphoramidites
remove protecting groups, cleave bead linker
purify
Similar chemistry used for RNA.
Glass Stepwise yield >99%. Bead Can use to make DNA/RNA up to 100 bases long.