Synthetic Nucleotides: Expanding Past the Four Nucleotide DNA “Alphabet” Sammy Zucker

The Discovery of 5-methylcytosine ● Supposed “fifth” nucleotide ● Created through the methylation of cytosine ● Illustrates the possibility of nucleotide modification dC 5-methylcytosine

The Discovery of 5-hydroxymethylcytosine ● “Sixth” nucleotide ● Found predominantly in brain and stem cells ● Cytosine with both a methyl and hydroxyl group ● Thought to play a role in demethylation dC 5-hydroxymethylcytosine

Unnatural DNA Dr. Floyd Romesberg has led the search for synthetic DNA base pairs through both chemical and biological approaches.

Optimized Polymerases The biological approach attempted to maximize the efficiency of the polymerase which replicated the unnatural DNA base pairs to aid in its replication.

Synthesized Nucleotides - Initial Efforts The chemical approach was the actual synthesis of unnatural nucleotides, and the selection of the most efficient of these nucleotides.

Synthesis of the d5SICS-dNaM Base Pair Out of many candidates, d5SICS and dMMO2 were found to make the most efficient pair, and were then further refined to the pair of d5SICS and dNaM.

Why is this pair efficient? The unnatural base pair is able to adapt a conformation within the polymerase similar to the dG-dC pairing.

In Vivo Replication of Synthetic DNA This year, the Romesberg lab was able to replicate DNA containing the synthetic nucleotides within an E.Coli cell.

Applications of Synthetic DNA The addition of an unnatural base pair vastly increases diversity, advances synthetic biology, while also having practical applications in many fields.

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