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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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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Aptamers

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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.

The DeRosa lab has recently published a proof-of-concept study where an aptamer was conjugated to a DTPA chelate.

Thrombin

• Thrombin is an enzyme in blood plasma that causes the clotting of blood by converting fibrinogen to fibrin.

• Blood clots can be very dangerous as they can break loose and move to other parts of 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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A 29 base long DNA aptamer that binds to thrombin in blood clots was isolated by Kubik et al. in 1997.(3)

This aptamer will form the basis of a study of contrast agents to determine the best system for imaging thrombin in serum.

MRI

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Magnetic Resonance Imaging Machine Gadolinium

Contrast Agent

SCN-DTPA

SCN-DOTA

A B

C

D

E

F

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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 administering 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.

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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Preparation of the conjugate

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- UV Vising the DNA was quantified using UV Vis - Sending the conjugates to mass spectrometry to

make sure the aptamer and chelate are together. 2023-04-12 Daphnée Dubouchet-Olsheski 8

R1 = the chelator (DTPA and DOTA)R2= the aptamer

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Results

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Results

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Results

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Results

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Results

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Results

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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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References and AcknowledgementsI 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, Gunther 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.