1. Development Of An Aptamer-Based
MRI Contrast Agent For Thrombin
Detection
Daphnée Dubouchet-Olsheski
under the supervision of
Erin McConnell, Maria DeRosa.
March something
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2. Outline
• Background Information
• Aptamers
• Thrombin
• MRI
• Introduction
• Project Goals
• Preparation of the Conjugate
• Results and Interpretations
• Relevant Application
• References and Acknowledgements
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3. Aptamers
The DeRosa lab has recently published a proof-of-concept
study where an aptamer was conjugated to a DTPA
chelate.
Aptamers are oligonucleic
acid or peptide molecules
that bind to a specific target
molecule.
Aptamers are single stranded DNA or
RNA sequences that fold into distinct
nanoscale shapes capable of binding
specifically to a target molecule.
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4. Thrombin
• Thrombin is an enzyme in blood A 29 base long DNA aptamer that binds
plasma that causes the clotting to thrombin in blood clots was isolated
of blood by converting fibrinogen by Kubik et al. in 1997.(3)
to fibrin.
This aptamer will form the basis of a
• Blood clots can be very
study of contrast agents to determine
dangerous as they can break
the best system for imaging thrombin in
loose and move to other parts of serum.
your body.
• MR imaging of thrombin could be
useful in the imaging and
isolation of blood clots
• The targeting of thrombin could
prove useful for precise MR
imaging of internal bleeding and
blood clotting processes.
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5. MRI
D
A B
Magnetic Resonance
Imaging Machine Gadolinium
SCN-DTPA E
C
F
Contrast Agent SCN-DOTA
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6. MRI
• Magnetic Resonance Imaging is a procedure used in hospitals to scan
patients and determine the severity of certain injuries. An MRI machine
uses a magnetic field and radio waves to create detailed images of the
body.
• Signal contrast in MR images depends on the “relaxation” of in vivo water,
which can be increased by administrating a contrast agent (CA)..
• Paramagnetic gadolinium(III) (Gd(III)). Is a contrast agent used in the MR
Imaging of Thrombin.
• The gadolinium increases the relaxation time of tissues.
• Gd(III) cannot be administered as a free ion because of its high toxicity.
• It is typically chelated with a compound such as die-thy-lene-triamine-
penta-acetic acid (DTPA) or 1,4,7,10-tetra-aza-cyclo-do-decane-1,4,7,10-
tetra-acetic acid (DOTA) for use as an MRI agent.
• Using nanotechnology and DNA synthesis we have been able to create
specific receptor molecules (Aptamers) that can target specific tissues
such as thrombin.
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7. Project Goal
• The development of a targeted MRI contrast agent could
enhance the diagnostic value of the obtained MR
images.
• In this study, the goal is to use synthetic receptors known
as aptamers to develop an MRI contrast agent that is
specific for the protein thrombin.
• This may allow for the precise imaging of blood clots.
• The hope to screen two series of contrast agents (DTPA
and DOTA) to find the best system for measuring
thrombin in serum.
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8. Preparation of the conjugate
R1 = the chelator
(DTPA and DOTA)
R2= the aptamer
The preparation of the conjugate
included:
- Synthesizing the DNA using the MerMade
Software
- Reacting the DNA columns with the chelate
(DTPA or DOTA)
- Recovering olingonucleotide from gel
- Desalting the DNA
- Pass the conjugate through a UV vis to make
sure the aptamer and chelate are together.
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9. Results
• The successful synthesis of the amino modified aptamer was
confirmed by mass spectrometry.
• By comparing the mass observed in the spectrum to the theoretical
mass of the aptamer-DTPA and aptamer-DOTA conjugates it was
obvious that the conjugate had not been synthesized.
• This meant that the DTPA and DOTA chealators did not bind to the
aptamers to form the aptamer-chelate conjugate.
Figure 2 and 3, results for mass spectrometry show that the mass of
the DNA-Chelate conjugates were far to low (where theoretical mass
was 10,000), suggesting that the chelates did not bind.
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10. Results
The yield and purity of the amino-modified aptamers was low.
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11. Results
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12. Results
The yield and purity of the aptamer-chelate conjugates, as well
as the Gd(III) loading must be high to ensure that these
conjugates can be prepared efficiently. Therefore the DNA was
run through a denaturing gel to separate the Gadolinium ions
from the DNA before attempting the aptamer-chelate reaction
again.
The DNA was reacted once more with the DTPA/DOTA chelate
to form the aptamer-chelate conjugate. This time the
conjugates showed better yield and purity.
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13. Results
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14. Results
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15. Results
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16. Results
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17. Relevant Application
• The targeting of thrombin could prove useful
for precise MR imaging of internal bleeding
and blood clotting processes.
• For instance, there is interest in MR imaging of
coronary thrombosis and pulmonary
embolism, circumventing the conventional,
much more invasive, angiography and
angioscopy procedures. (4)
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18. References and Acknowledgements
I would like to thank Carleton University for providing the DNA synthesizer, the gel
electrophoresis setup, the UV-Vis spectrometer and access to the 1.5 T MRI (Ottawa
Hospital) through a collaboration with Dr. Eve Tsai.
References:
(1) Caravan P, Ellison JJ, McMurry TJ, Lauffer RB “Gadolinium(III) chelates as MRI
contrast agents: structure, dynamics, and applications.”Chem. Rev. 1999, 99, 2293
2352.
(2) Bernard, E.D.; Beking, M. A.; Rajamanickam, K.; Tsai, E. C.; DeRosa, M. C.
“Target binding improves relaxivity in aptamer–gadolinium conjugates” J. Biol. Inorg.
Chem., 2012 DOI: 10.1007/s00775-012-0930-z
(3) Tasset, D. M.; Kubik, M. F.; Steiner, W. “Oligonucleotide inhibitors of human
thrombin that bind distinct epitopes” J. Mol. Biol. 1997, 272, 688-698.
(4) Spuentrup E, Buecker A, Katoh M, Wiethoff AJ, Parsons Jr EC, Botnar RM,
Weisskoff RM, Graham PB, Manning WJ, Günther RW “Molecular Magnetic Resonance
Imaging of Atrial Clots in a Swine Model” Circulation 2005, 111, 1377-1382.
(5) Munshi KN, Dey AK “Absorptiometric study of the chelates formed between the
lanthanoids and xylenol orange” Microchim. Acta 1968, 56, 1059-1065.
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