MCB102 Fall 2024 Lecture 2: Nucleic Acid Chemistry PDF

Lecture 2: Nucleic Acid Chemistry What chemical reactions occur to nucleic acids? How do these reactions help or hurt the transmission of genetic information?...

Lecture 2: Nucleic Acid Chemistry What chemical reactions occur to nucleic acids? How do these reactions help or hurt the transmission of genetic information? How do we learn the sequence of a DNA molecule? Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 1   Lecture outline Common Chemical Reactions Making and Breaking Phosphoester Bonds Sequencing of Nucleic Acids Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 2  Our abstracted information carrier has a physical layer; these molecules can partake in chemical reactions Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 3  Deamination is a common spontaneous reaction Most prevalent Also in base editing deamination = spontaneous loss of exocyclic amino groups Important in evolution deamination of cytosine to uracil = ~100 events/day recognized as foreign in DNA and removed Notable in base almost certainly why DNA editing contains thymine rather than uracil Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 4  Cytosine deamination can lead to mutation G≡C G=U Cytosine Uracil Spontaneous cytosine deamination: G≡C A=U Rate ~5×10-13 s-1 = 1 / 50000 years ~3 billion cytosines / cell After C→U, DNA replication inserts A 5 C→U / cell / MCB102 lecture Mutation: C→U and G→A Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 5  Deamination and distinguishing thymine vs uracil Uracil in DNA Repair ≠ Cytosine Uracil Thymine Spontaneous cytosine deamination: Thymine methyl distinguishes it from C→U Uracil in DNA ef ciently removed and repaired Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 6 fi  Ancient DNA highlight (2022 Nobel Prize for former Berkeley research fellow Svante Paabo!) Cytosine Uracil Spontaneous cytosine deamination: 50,000 year timescale for ancient DNA sequencing High frequency C→U changes in ancient DNA Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 7  Enzymatic cytosine deamination Cytosine Uracil Enzymatic cytosine deamination: Activation-induced deaminase: mutates antibody genes in B lymphocytes APOBEC enzymes: mRNA editing by C→U changes encoded protein in some genes anti-viral “editing” C→U Related to a type of genome editing called base editing Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 8  Adenine can also be deaminated Donor Acceptor Adenine Hypoxanthine Guanine (Base in inosine) Adenine deamination: Hypoxanthine nucleoside is called inosine Adenosine deaminase: A→I editing in mRNA Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 9  Cytosine methylation 5-Methylcytosine: Analogous to thymine Uracil Cytosine No change to H-bonding No change to base pairing Thymine 5-methylcytosine Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 10  More on cytosine and methylation Cytosine methylation: S-adenosylmethionine source of methyl Bacterial cytosine (and adenine) methyl: Mark “self” DNA versus bacteriophage DNA Eukaryotic cytosine methylation (5MeC): Cytosine Occurs at CpG sites Important in controlling gene expression Non-base-pairing information in DNA 5-methylcytosine Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 11  Methylation and deamination Uracil in DNA Repair Cytosine Uracil Thymine in DNA 5-MeCytosine Thymine What’s the problem? Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 12  Lecture outline Common Chemical Reactions Making and Breaking Phosphoester Bonds Sequencing of Nucleic Acids Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 13  Transesterification Cutting, extending, and joining nucleic acid sequences Essentially one chemistry for all backbone changes: Transesteri cation Nucleophilic attack on a phosphate Pentavalent phosphate intermediate or transition state Resolved by a different group leaving the phosphate Intermediate Nucleophile Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 14  RNA self-cleavage - 2 OH is a built-in nucleophile The 2’-OH on RNA can act as a nucleophile 25º C, neutral pH: 4-year half-life Heat, alkali, cations accelerate this Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 15 ’  Enzymes The kinds of enzymes that make or break backbone bonds Named according to reactions they carry out Nuclease Breaks apart DNA or RNA backbone Hydrolysis — transesteri cation onto water Exonuclease cuts only at the ends of molecules Endonuclease cuts in the middle of molecules Many speci cities — DNA vs RNA, ss vs ds, sequences Polymerase Joins nucleotides into nucleic acid polymers Usually adds to the end of existing DNA or RNA Always adds at the 3’ end, on a 3’-OH Uses nucleotide 5-triphosphates: NTPs or dNTPs Ligase Joins 3’ end of one polymer to 5’ end of another Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 16 fi fi  Restriction endonucleases: enzymes that cut DNA Cuts in the middle of dsDNA at a speci c “recognition sequence” 4 - 8 bases long Cuts each strand Often cuts only unmethylated DNA Bacterial genome is methylated Virus (bacteriophage) DNA is not methylated Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 17 fi  Restriction endonucleases: enzymes that cut DNA HO:⊖ 5’ 5’ P P P P P P P P P P P P P P P G A G A A T T C T A C C C T C T T A A G A T G G 3’ P P P P P P P P P P P P 3’ 1’ (deoxy-)ribose HO:⊖ G A 5’ 5’ P P P P P P P P P P P P OH A A T T C T A C C G A G G A T G G C T C T T A A OH P P P P P P P P P P P P Lecture 02: Nucleic Acids: Chemistry and Reactions 3’ MCB102 Fall 2024 18  Polymerases synthesizes nucleic acid polymers Adding nucleotides to DNA (or RNA): 3’-OH activated for nucleophilic attack on the rst (α) phosphate of dNTP (or NTP) with 5’-triphosphate Inorganic pyrophosphate (PPi) leaves Pyrophosphate typically hydrolyzed, which improves thermodynamics Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 19 fi  Polymerases in detail DNA and RNA Polymerases: 3’ end of primer strand grows (5’ → 3’ on synthesized strand) Base pairing of dNTP with template nucleotide Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 20  Lecture outline Common Chemical Reactions Making and Breaking Phosphoester Bonds Sequencing of Nucleic Acids Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 21  DNA Sequencing Bandwidth is Improving Exponentially >4 methods in use; we ll talk about 3 Sanger (Old school): 1 read of 1000 bp for $10 Illumina (short read): 108 reads of 100 bp for $1000 PacBio (long read): 107 reads of 10000+ bp for $1000 Nanopore (not covered here) Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 22 ’ ‘ ’  If you are interested in details, check out this lecture from Prof. Eric Chow at UCSF https://www.youtube.com/watch?v=mI0Fo9kaWqo Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 23  Sanger Sequencing using dideoxynucleotides 3’-H dideoxynucleotide No OH to serve as a nucleophile Chain terminator Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 24  Sequencing with ddNTPs 5’-GAG-3’ 3’-CTCTTAAGATCG-5’ DNA Polymerase dATP, dCTP, dGTP dideoxyTTP 5’-GAGA-3’ 5’-GAGAAT-3’ 5’-GAGAA-3’ 3’-CTCTTAAGATCG-5’ Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 25  Sequencing with ddNTPs 5’-GAG-3’ 3’-CTCTTAAGATCG-5’ DNA Polymerase DNA Polymerase dATP, dCTP, dGTP dATP, dGTP, dTTP dideoxyTTP dideoxyCTP 5’-GAGAAT-3’ 5’-GAGAATTC-3’ 3’-CTCTTAAGATCG-5’ 3’-CTCTTAAGATCG-5’ Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 26  Thought experiment on adding ddNTPs to polymerase reaction 5’-GAG-3’ 3’-CTCTTAAGATCG-5’ ⊖ ddATP ddCTP ddGTP ddTTP 12 5’-GAGAATTCTAG-3’ 5’-GAGAATTC-3’ 5’-GAGAAT-3’ … 4 5’-GAGA-3’ 3 2 1 ⊕ Gel Electrophoresis Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 27  Now, consider adding in a small amount of ddNTPs 5’-GAG-3’ 3’-CTCTTAAGATGG-5’ DNA Polymerase dATP, dCTP, dGTP 5’-GAGAAT-3’ a small amount 3’-CTCTTAAGATGG-5’ dideoxyTTP or mostly dTTP 5’-GAGAAT-3’ 3’-CTCTTAAGATGG-5’ Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 28  Now, consider adding in a small amount of ddNTPs 5’-GAG-3’ 3’-CTCTTAAGATGG-5’ DNA Polymerase dATP, dCTP, dGTP 5’-GAGAAT-3’ a small amount 3’-CTCTTAAGATGG-5’ dideoxyTTP mostly dTTP 5’-GAGAATT-3’ 3’-CTCTTAAGATGG-5’ or 5’-GAGAATT-3’ 3’-CTCTTAAGATGG-5’ Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 29  Now, consider adding in a small amount of ddNTPs 5’-GAG-3’ 3’-CTCTTAAGATGG-5’ DNA Polymerase dATP, dCTP, dGTP 5’-GAGAAT-3’ a small amount 3’-CTCTTAAGATGG-5’ dideoxyTTP mostly dTTP 5’-GAGAATT-3’ 3’-CTCTTAAGATGG-5’ 5’-GAGAATTCT-3’ 3’-CTCTTAAGATGG-5’ Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 30  Sanger sequencing uses fluorescent ddNTPs and capillary electrophoresis “Modern” Sanger sequencing One reaction Different dyes per ddNTP Simultaneously terminate and label DNA Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 31  Sanger sequencing uses fluorescent ddNTPs and capillary electrophoresis Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 32  DNA sequencing is changing rapidly: Reversible Terminator Sequencing aka Illumina sequencing Uses four different modi ed deoxynucleotides (A, T, G, and C), each with a particular uorescent label and a 3′ blocking group Advantage: millions to billions of molecules are sequenced Disadvantage: reads are short, e.g. ~150 base pairs Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 33 fl fi  Illumina bandwidth is possible because sequencing occurs at small physical scale on a miscroscope Time in cycles Each dot is a small cluster of many of the same DNA molecules being sequenced Flow cell Example reversible terminator Fluorescent, zoomed-in view of owcell Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 34 fl  DNA sequencing is changing rapidly: Single Molecule Sequencing aka PacBio (or SMRT) Advantage: single molecule processisivity allows read length averages >10,000 bp which can be VERY helpful in many cases Disadvantages: physical complexity of ow cell results in lower throughput (~10 millions reads) and higher cost Lecture 02: Nucleic Acids: Chemistry and Reactions MCB102 Fall 2024 35 fl