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

3. 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,
Copyright ©2006 by the National Academy of Sciences

4. 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

5. 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

6. 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)

7. 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?

8. 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

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

10. 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

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

12. 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

13. 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?

14. Synthesis of GNA

15. 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

16. 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

17. Part II: The Protein World

18. 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?

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

20. 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

21. Results:
Some insoluble material: likely a cyanide-aldehyde polymer
Aqueous residue showed that % 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 

22. 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!

23. Proteinogenic Amino Acids:

24. 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)

25. 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

26. After drying, paper is exposed to ninhydrin:

27. Ninhydrin reacts with amino acids via imine formation:

28. 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

29. 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

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