Angewandte
Chemie
DOI: 10.1002/anie.200906712
Imaging Techniques
A Secreted Enzyme Reporter System for MRI**
Gil G. Westmeyer, Yves Durocher, and Alan Jasanoff*
An important goal in modern biology is to understand how
molecular processes commonly studied at the cellular level
give rise to physiological functions in complex tissues and
organisms. Non-invasive imaging of gene-expression patterns
in whole animals could provide information critical to this
end, but current methods lack sensitivity and spatiotemporal
precision. Enzymatic reporter systems detectable by magnetic
resonance imaging (MRI) address these limitations by
combining the relatively high spatial and temporal resolution
of MRI with the ability of each genetically expressed enzyme
to generate many MRI-detectable product molecules.[1,2]
A
challenge with the imaging-based detection of some of the
most popular reporter enzymes is the need to deliver MRI
probes to their sites of action within cells. Herein we describe
a new reporter-gene system for MRI that relieves this
problem by harnessing an extracellular enzyme, the mamma-
lian secreted alkaline phosphatase (SEAP).
SEAP is a truncated, secreted variant of placental alkaline
phosphatase (PLAP). It is widely used as a stable and
heterologously expressible reporter enzyme in conjunction
with optically absorbent, fluorescent, or luminescent sub-
strates.[3] For the optimal detection of SEAP activity by MRI,
we modified an existing sensor for adenosine (Ado), which is
produced by SEAP’s hydrolysis of phosphorylated adenosine
derivatives. In this system, the reporter enzyme is therefore
detected through its generation of product molecules, as
opposed to its direct action on an MRI contrast agent. The
process is reversed upon the removal or degradation of Ado,
is nondestructive to the Ado sensor, and is relatively fast,
because SEAP substrates can be used at concentrations well
above their Km values without affecting the background MRI
signal (Figure 1a).
Figure 1. SEAP-based reporter-gene system for MRI. a) Secreted alka-
line phosphatase (SEAP, red) is expressed and secreted from genet-
ically modified cells (left). Extracellular SEAP cleaves 2’-AMP or a
related substrate to generate adenosine (Ado, plus inorganic phos-
phate, Pi). Ado is then detected by a SPIO-based MRI sensor (right)
actuated by an adenosine-binding aptamer (dark blue). Sensing can be
reversed by the destruction or removal of Ado. b) Relative changes in
the T2 relaxation rate reported as a function of Ado concentration from
prototype (light blue) and optimized (dark blue) SPIO-based Ado
sensors. Relative DR2 =[(R2)obsÀ(R2)min]/[(R2)maxÀ(R2)min], in which
(R2)obs is the R2 value observed at each Ado concentration, and (R2)max
and (R2)min denote the maximal and minimal recorded R2 values.
Titration curves were fitted to a Hill equation to yield EC50 values of
(1.0Æ0.2) mm and (91Æ14) mm for the Ado responses of the proto-
type and optimized sensors, respectively. Error bars (standard error of
the mean, SEM) for some data points are obscured by symbols in the
graph.
The Ado sensor that we used is actuated by an Ado-
binding DNA aptamer.[4] In the absence of Ado at saturating
concentrations, this aptamer cross-links superparamagnetic
iron oxide nanoparticles (SPIOs) modified with reverse-
complementary DNA segments.[5,6] Ado-dependent disaggre-
[*] Dr. G. G. Westmeyer, Prof. A. Jasanoff
Departments of Biological Engineering, Brain & Cognitive Sciences,
and Nuclear Science & Engineering
gation of the functionalized SPIOs modulates their ability to
create contrast in T2-weighted MRI scans. Experimental[7]
and theoretical[8] studies showed that if nanoparticles with a
diameter greater than about 50 nm are used, disaggregation
accompanies an increase in the T2 relaxation rate (R2 = 1/T2);
the use of smaller SPIOs leads to the opposite change in
R2.[8–10] A prototypical Ado sensor formed from SPIOs with a
mean diameter of (106 Æ 1) nm, as measured by dynamic light
scattering (DLS), showed a 50% change in relaxation rate
(R2 = 1/T2) at an Ado concentration (EC50) of (1.0 Æ 0.2) mm
(Figure 1b). To improve on this apparent affinity, we used
hybridization rules to predict thermodynamically favorable
Massachusetts Institute of Technology
150 Albany Street, NW14–2213, Cambridge, MA 02139 (USA)
Fax: (+1)617-253-0760
E-mail: jasanoff@mit.edu
Dr. Y. Durocher
NRC Biotechnology Research Institute
6100 Royalmount Avenue, Montrꢀal, Quebec, H4P 2R2 (Canada)
[**] We thank the Raymond and Beverley Sackler Foundation for their
generous support. Additional funding was provided by NIH grant
DP2-OD002114 to A.J. MRI=magnetic resonance imaging.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2010, 49, 3909 –3911
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3909