Directed Evolution of a Magnetic Resonance Imaging Contrast

Agent for Noninvasive Imaging of Dopamine

Mikhail G. Shapiro, Gil G Westermeyer, Philip A Romero, Jerry O Szablowski & Alan Jasanoff -Volume 28 Number 3 March 2010

A R T I C L E S

Overview

• OBJECTIVE: Develop an MRI contrast agent for sensing dopamine-related activity in the brain

• Dopamine plays a crucial role in learning and motor coordination; dysfunction underlies addiction

• Current techniques to measure dopamine activity are either invasive/ PET procedures with low spatial and temporal resolution

• SOLUTION: Engineer a paramagnetic protein, flavocytochrome BM3, to selectively bind to dopamine- 1% contrast in vivo

Background on MRI

• Magnetic Resonance Imaging is non-invasive imaging technique used

• Gradient magnetic field gradients induce spins on H-atoms.

• Time taken to return to equilibrium spin –> T1 relaxation time

• Relaxivity – measure of contrast (r1, 1/s)

Fig. 1: MRI Machine Fig. 2: MRI Contrast Images

Heme Iron Interaction Dynamics

• Heme iron with H20 promotes r1

• Native ligand is arachidonic acid (AA)

• Ligand induces decrease in relaxivity r1 contrast on MRI

• Can we select for dopamine ?

Fig. 3: BM3 Ligand Binding

Directed Evolution Process

• Start with WT gene housed pCWOri vector

• Error-Prone PCR with Taq polymerase, MnCl2

• 1 or 2 mutations/ gene transformed into E.Coli • I366V into BM3h variant to improve protein thermostability (via overlap

extension PCR)

Fig. 4: Library Generation Schematic

Screening and Results

Ligand-induced 50 % !

• Two mutants : BM3h- 8C8, BM3h-B7 selected

• Screening performed via absorbance assay

• Confirmed via MRI contrast imaging

Testing of Variants

In Vitro In Vivo

• PC12 cells- derived from adrenal gland of rats-in F12-K

• Dopamine release only in

presence of extracellular K+

• Brain of anesthetized rats

Conclusions

• Created MRI contrast agents sensitive to neurotransmitter dopamine – heme domain of flavocytochrome BM3

• Developed novel screening methodology - ligand induced decrease in relaxivity under MRI -and optical characterization

• Evolved specificity of BM3h-based sensors away from natural ligand and toward dopamine via error-prone PCR.

• Introduced mutation I366V by site-directed mutagenesis to enhance thermostability and tolerance to further mutation

• Performed In Vitro (PC12) and In Vivo (live animal) testing of efficacy of developed sensors

• General paradigm for development of molecular probes for MRI with tunable ligand-binding and catalytic properties