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

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

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. T m = 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” T m 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 (T m = + 3°C) & with DNA (T m = + 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

Duplex formation –PNA-DNA T m = 68.8 °C (~20 °C higher than DNA-DNA!) –PNA-RNA T m = 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 T m = 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