http://www.scq.ubc.ca/

Sequencing by Synthesis Newer method to sequence whole genomes Uses allyl protecting group:

Ju, Jingyue et al. (2006) Proc. Natl. Acad. Sci. USA 103, 19635-19640 Sequencing by Synthesis Ju, Jingyue et al. (2006) Proc. Natl. Acad. Sci. USA 103, 19635-19640 Copyright ©2006 by the National Academy of Sciences

DNA/RNA Analogues There have been several recent reports of the modification of oligonucleotides Modifications have included: Altering nitrogenous base structure Employing different sugar structures Modifying the backbone Or in combination

Why make oligo analogues? Structure/activity relationships (i.e., catalytic versatility) Antisense technology Insight into evolutionary process? Synthetic oligos can be designed to bind with itself, DNA, RNA or all of the above Effects: H-bonding (base-pairing) Other types of interactions (i.e. Van de Waals) Overall shape (i.e., double helix? Hairpin loop?) How do we examine interactions? Melting temp. Tm = 40.5 °C (DNA), 42.5 °C (RNA) NMR (as well other spectroscopic methods) Xray crystallography Calculations

Antisense Technology Employs a synthetic oligo that is complementary (antisense) to an mRNA sequence of interest One of Two main effects can occur: Translation arrest (no protein) Recruits RNAase that degrades mRNA Potential for therapeutic use Vitravene (acts on CMV virus)

Unnatural Base Pairs Recall the natural base pairing (Watson-Crick) in DNA: If we change the number of or location of donor/acceptor groups, what interactions can occur?

For example: Expand the “genetic alphabet” Tm measurements vary dramatically due to changes in H-bonding properties & hydrophobicity DNA polymerase found to work with some modified bases (oligos) Such structures would be unlikely to form under prebiotic conditions

Employing Novel Sugars Some examples: 5- vs 6- membered ring Location of phosphodiester bond Hydroxyl groups (# & stereochemistry) Position of base

Adopts chair conformation in oligo Forms helical duplex with RNA (Tm = + 3°C) & with DNA (Tm = + 3°C) Adding more hydroxyl groups, increases affinity for RNA Also increases thermal stability

Modification of the Backbone Peptide Nucleic Acid (PNA) Aminoethyl glycine units linked by a peptide bond achiral

Did a PNA molecule precede RNA? Duplex formation PNA-DNA Tm = 68.8 °C (~20 °C higher than DNA-DNA!) PNA-RNA Tm = 72.2 °C  PNA has the recognition properties of DNA (can carry genetic information) & has a higher stability than RNA and DNA Stability? Lack of phosphate groups → neutral backbone  no electrostatic repulsion! Not recognized by proteases Did a PNA molecule precede RNA? Has demonstrated numerous forms: hairpins, triplexes, etc Simple in structure (i.e., achiral) & stable Amino building blocks in primordial soup

Another Modified Nucleic Acid - GNA Glycol nucleic acid Can nucleic acid be made with such a simple sugar? Is a ring structure important? Minimum # of carbons?

Synthesis of GNA

Tested duplex formation: Duplex formation Tm = 63 °C (22 ° higher than same DNA or RNA sequence!)  GNA more stable than DNA! Demonstrates that cyclic sugar not necessary! Was ribose in fact the sugar in the first pre-biotic nucleic acids? Is GNA a pre-RNA candidate? A glycerol derivative, which is related to triose Recall, we looked at 3C sugars (i.e., glyceraldehyde) in relation to the prebiotic formation of sugars

An example of chemical biology: Uses biological concepts, DNA structure Uses chemical ideas → conformation & functional groups Uses chemical synthesis principles Makes a non-natural molecule with novel properties Relates those properties to the natural system Problem: Still difficult to predict and analyze single-stranded oligonucleotide structures

Part II: The Protein World

We’ve seen how early catalysis by e. g We’ve seen how early catalysis by e.g. clay, leads to synthesis of more complex structures These in turn led to catalysis of specific reactions, eventually leading to proteins taking over as the “normal” catalysts RNA world  Protein world Questions How were amino acids first formed? Origin of homochirality: relationship between D-sugars & L-amino acids? How did amino acids condense to give peptides in the prebiotic world? Is this related to chemical peptide synthesis Ribosomal peptide synthesis  relationships? Can this knowledge be used to evolve better synthetic strategies?

How were Amino Acids Formed? The Urey-Miller Experiment:

The Urey-Miller Experiment CH4, NH3 & H2 atmosphere + H2O Water is heated to induce evaporation Vapor reaches gas & sparks are then fired through atmosphere (simulates lightning) Atmosphere is then cooled Water and organic compounds are trapped Continued experiment for 7 d, then analyzed residue

Results: Some insoluble material: likely a cyanide-aldehyde polymer Aqueous residue showed that 10 -15% of carbon had been converted to organic compounds (including amino acids) Glycine (R=H) was found to be most abundant (least C-C bond forming reactions needed) 12 of the other proteinogenic amino acids (20 in modern cells) were formed: These were  amino acids (C relative to C=O) Note: there are >> 400 naturally occurring amino acids, including both enantiomers, -amino acids, etc 

Gamma-aminobutyric acid A  amino acid For example: Gamma-aminobutyric acid A  amino acid The amino acids used for protein synthesis are always L and the  stereocentre (except gly) is has S configuration D amino acids do occur & are often found in nonribosomal peptides Constructed on a nonribosomal peptide synthase (NRPS) Found primarily in bacteria and fungi More on these later!

Proteinogenic Amino Acids:

Neutral Amino Acids H+ donors (acidic) H+ acceptors (basic) Alkyl (Ala, Ile) The amides (Asn, Gln) Alcohol (Ser, Thr) Thiol (Cys) Thioether (Met) H+ donors (acidic) Phenol (Tyr) Acids (Asp, Glu) H+ acceptors (basic) Amines (Lys, His, Arg)

How to show amino acids are formed in the Urey-Miller Experiment? Ninhydrin detection of amino acids separated by electrophoresis: The more –ve charge on the AA at a given buffer pH, the faster it moves towards the cathode (+) +ve charged AAs move toward the anode

After drying, paper is exposed to ninhydrin:

Ninhydrin reacts with amino acids via imine formation:

Cannot distinguish between amino acids This condensation product has an extended conjugated system (how many resonance forms can you draw?) It absorbs light in the visible region (recall that in UV/VIS spectroscopy, max depends on the length of the conjugated system)  if we spray ninhydrin on the paper chromatogram (or on a TLC plate), purple spots develop Cannot distinguish between amino acids Alternatively, can separate amino acids by ion exchange chromatography Separates based on charge Derivatize with ninhydrin as eluant comes off the column

Subsequent studies of Urey-Miller Exp’t also showed: Rapid initial formation of HCN & aldehydes This was followed by a decrease of these products as amino acids were slowly formed Hypothesized that amino acids were being formed via a previously known method, the Strecker synthesis Strecker Synthesis: Developed by Strecker in 1850 to give racemic amino acids Can still be found in use, with some modifications

Strecker Synthesis: mechanism Was this how amino acids formed in the prebiotic world?