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 >
T
1
relaxation time
Relaxivity measure of
contrast (r
1, 1/s
)



Fig. 1: MRI Machine Fig. 2: MRI Contrast Images
Heme Iron Interaction Dynamics
Heme iron with H
2
0 promotes r
1


Native ligand is arachidonic acid (AA)

Ligand induces decrease in relaxivity
r
1
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, MnCl
2

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