Slide Presentation

Development Of An Aptamer-Based MRI Contrast Agent For Thrombin

Detection

Daphnée Dubouchet-Olsheski

under the supervision of

Erin McConnell, Maria DeRosa.

March something

2023-04-08 Daphnée Dubouchet-Olsheski 1

Outline

• Background Information• Aptamers• Thrombin• MRI

• Introduction • Project Goals • Preparation of the Conjugate

• Results and Interpretations• Relevant Application• References and Acknowledgements


Aptamers


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.


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


Magnetic Resonance Imaging Machine Gadolinium

Contrast Agent

SCN-DTPA

SCN-DOTA

A B

C

D

E

F


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.

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.


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- Pass the conjugate through a UV vis to make

sure the aptamer and chelate are together.


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


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.


Results

The yield and purity of the amino-modified aptamers was low.


Results


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.


Results


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)


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.

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