Photocage Project

Introduction

Amino acids not included in the native 20 are commonly known as non-canonical amino acids (ncAAs). These ncAAs have unique chemistry that can provide very useful functionality not normally present in an organism. For our purposes, ncAAs were used to prevent protein functionality until spatially and/or temporally triggered.

Background

This summer our team has been working on re-creating a light-activatable T7 RNA Polymerase (RNAP) for a method of non-invasive, spacio-temporal control of protein expression. T7 RNAP was our enzyme of choice for this project due to the presence of a tyrosine residue in the active site of the polymerase. By recoding this tyrosine residue, ortho-nitrobenzyl tyrosine was incorporated into the active site thus acting as a molecular cage. T7 RNAP is only functional when exposed to a certain wavelength of light that cleaves a molecular cage from the polymerase’s active site. In our experiments, ortho-nitrobenzyl tyrosine (ONBY) was used as our photocaged ncAA. ONBY was used because once the ONB group is cleaved off, the ncAA functions as a normal tyrosine. This proved to be particularly useful because T7 polymerase has a tyrosine residue in its active site that is necessary for proper function of the protein. Once de-caged, the polymerase is free to transcribe sequences that are preceded by a T7 promoter. GFP was used as a reporter to analyze and optimize each construct for spacio-temporal specificity. In addition, GFP was used to examine the effect of a certain non-canonical amino acid on fluorescence when placed in the fluorophore.

Data