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Jonathan Sczepanski, PhD

Research Associate
Nov 2010 – present
Ph.D., 2010, Johns Hopkins University
Research: Synthetic genetic systems
E-mail: jsczepa@scripps.edu



Sczepanski Figure 1Split-and-pool synthesis: The R3C ligase ribozyme was converted to a format whereby two enzymes catalyze each other’s formation, enabling their self-sustained exponential amplification. The replicating ribozymes can mutate and evolve, providing a working model of a genetic system. Inheritance of genetic information in this system system relies on two region of Watson-Crick pairing between the catalytic partners. These are the “genotypes” regions, each of which can encode a corresponding sequence, or “phenotype”, that brings about the catalytic function of the enzyme. As in nature, the relationship between the genotype and phenotype is described by a genetic code. In this system, however, the researcher chooses the code.

In order to prepare diverse populations of these cross-replicating enzymes, a novel split-and-pool technique was devised using solid-phase DNA synthesis. The genotype and phenotype portions of the molecule are synthesized in tandem on two different arms of the same DNA molecule by employing three orthogonal phosphoramidite protecting groups. Following synthesis, the DNA molecules are enzymatically circularized, which physically links the genotype and phenotype on a molecule-by-molecule basis. PCR amplification and transcription of these DNA templates produces the corresponding populations of RNA.

With the goal of evolving novel function in a self-sustained system, this synthetic strategy is now being used to prepare new populations of cross-replicating enzymes in order to study how the choice of genetic code influences evolution.

Sczepanski Figure 2Molecular evolution and epigenetics: Epigenetics is the study of gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. Examples include DNA methylation and histone post-translational modification. Unfortunately, tool for studying individual epigenetic modifications are quite limited. In vitro evolution offers an attractive method for developing these tools. With a goal of studying the mechanistic role of individual histone acetylation events in cells, a selection strategy has been devised to evolve ribozymes capable of histone acetylation. Histone H4 lysine 16 was chosen as the initial target due to this residues potent influence on the state of chromatin compaction. A selection to evolve this histone acetyltransferase (HAT) ribozyme is now underway.

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