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 pyrimidines purines pyrimidine purine 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..