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. 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.

Background

The tyrosine residue (Tyr639) that was replaced by ONBY lies on a crucial position, called the O-helix, that blocks incoming nucleotides from reaching the active site. The Tyr639 in the O-helix is responsible for two critical roles roles in polymerization. First, the tyrosyl-OH coordinates Mg2+ to interact with the 2’-OH of the incoming nucleotide to assure discrimination between deoxyribose and ribose substrates. Second, the Tyr639 moves the newly synthesized RNA out of the catalytic site and prepares for the next NTP to be inserted. These roles were determined to be critical through mutational analysis. Polymerases that lacked the Tyr639 residue resulted in a loss of function. Furthermore, because of the bulky nature of ONBY, its incorporation at the 639 position blocks incoming nucleotides from reacting in the active site. This group can be removed by brief irradiation by non-phototoxic UV light, which would restore RNAP activity. In order to introduce the photocaged tyrosine into the cell, a synthetase/tRNA pair that is specific for ONBY must be transformed into the bacteria. In order to do this, a Methanococcus jannaschii synthetase/tRNA pair was evolved in order to incorporate ONBY, but not any of the other 20 natural amino acids. In order to evolve the pair, six residues (Tyr 32, Leu 65, Phe 108, Gln 109, Asp 158, and Leu 162) on the synthetase were randomized and selected for its ability to selectively incorporate ONBY. The following are the specific changes made to the amino acid sequence of the synthetase: Tyr 32→Gly 32, Leu 65→Gly 65, Phe 108→Glu 108, Asp 158→Ser 158, and Leu 162→Glu 162. The Asp 158→Ser 158 and Tyr 32→Gly 32 mutations were likely involved in the loss of hydrogen bonds with the natural substrate, which would disfavor binding to natural tyrosine. On the other hand, the Tyr 32→Gly 32 and Leu 65→Gly 65 mutations likely increase the size of the substrate-binding pocket to accommodate the bulky o-nitrobenzyl group.

Experimental Methods

Note: I will elaborate in more words in the following sections

Preparation & Growth of Cultures

• Picked Colonies
  • PC 1: pQE/ONBY
  • PC 2: pQE/ONBY
  • PC 3: MJH117/ONBY
  • PC 4: MJH117/ONBY
  • PC 5: aT7/ONBY
  • PC 6: aT7/ONBY
    • Antibiotics for PC 1-4: Zeo, Kan, Gent
    • Antibiotics for PC 5-6: Zeo, Crb, Gent

• Conditions for each picked colony
  • (-) IPTG, (-) ONBY
  • (+) IPTG, (-) ONBY
  • (+) IPTG, (+) ONBY

• Inoculate each tube with designated PC; add 1 uL of cells into each tube
• Grow cultures for 2 hours
• Add IPTG to all (+) IPTG culture tubes
• Grow another 2-4 hours

Decaging ortho-nitrobenzyl tyrosine

• Take cultures out and irradiate with 365 nm light
  • Place all (+) IPTG (+) ONBY cells into wells of a 12-well plate
  • Place (+) IPTG (-) ONBY of PC 1, 3, 5 into wells
  • Place (-) IPTG (-) ONBY of PC 3 & 4 to use as a control for growth without ONBY (*this will allow us to see how much the ONBY solution decreases growth rate of cells)
• Place negative control into 12 well plate
  • Irradiate using handheld blacklight placed directly over the wells; irradiate cells and take out at every time point (0 min, 1 min, 5 min, 15 min, 30 min) and transfer to wells in a 96 well plate to test fluorescence

Measuring Fluorescence

NEED TO EDIT: THIS WAS CUT AND PASTED FROM A PAPER WE WROTE. BELOW: BETTER IMAGE resolution

Results