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Development Of An Aptamer-Based MRI Contrast Agent For Thrombin

Detection

Daphnée Dubouchet-Olsheski

under the supervision of

Erin McConnell, Maria DeRosa.

March something

2023-04-12 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 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 first phase of the project will be to synthesize and purify the aptamer-chelate conjugate.

• If time permits the second phase of the project, will involve screen two series of contrast agents (DTPA and DOTA) to find the best system for measuring thrombin in human serum.

• An MRI contrast agent can be made more specific for a certain protein by conjugating it to an aptamer. A practical MRI contrast agent for imaging thrombin and blood clots can be developed using aptamer technology.


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

make sure the aptamer and chelate are together.


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


Results

Figure 3: PAGE purification. Left : aptamer-DOTA conjugate. Right : aptamer-DTPA conjugate.


Figure 3: PAGE purification.

• The thick, dark bands show that the DNA was successfully synthesized.

• The bands containing DNA were cut out and the DNA-chelate conjugates were extracted and desalted to purify the sample of extra salts and urea.

• Subsequently the DNA was quantified by UV-Vis Spectroscopy.


Results

Figure 4: UV-Vis absorbance spectrum of DNA for quantification of the DNA-chelate conjugates.


Figure 4: UV-Vis absorbance spectrum of DNA for quantification of the DNA-chelate

conjugates. • The presence of DNA is indicated by a peak at ~260 nm (known value) as seen in

figure 4.

• Using Beer lamberts law the amount of DNA was quantified in nano moles based on the absorbance of the sample.

• The aptamer-DOTA conjugate was quantified at 296nmol and the aptamer-DTPA conjugate was quantified at 193 nmol.

• 3 nmol of each aptamer-chelate conjugate were sent out for mass spectrometry to see if the reaction worked.

• After the quantification of the aptamer-chelate conjugates the amount of Gd(III) to react could be calculated since the ratio of Gd(III) to aptamer-chelate conjugate should be 1:1.

2023-04-12 13

Results

Daphnée Dubouchet-Olsheski

Figure 5: Calibration curve from xylenol orange test.

Figure 6: Eppendorf tubes containing Xylenol Orange test reactions.


Figure 5: Calibration curve from xylenol orange test.Figure 6: Eppendorf tubes containing xylenol orange

test reactions.

• The xylenol orange test created a curve/function to determine the concentration of Gd(III) reacted with the conjugate (see figure 5).

• The absorbance ratio of the flowthrough (FT) from the Gd(III) incubation was compared with known concentrations of Gd(III) as represented in figure 6 and 7.


Results

Figure 7: UV-Vis absorbance spectrum of xylenol orange test calibration curve.


Figure 7: UV-Vis absorbance spectrum of xylenol orange test calibration curve.

• The xylenol orange test showed that the loading efficiencies of the chelates were less than 80%.

• For the loading efficiency to be higher more Gd(III) will be added.


Results

Figure 8: Mass spectrometry results


Results

Figure 8: Mass spectrometry results


Figure 8: Mass spectrometry results • To confirm that the aptamer-chelate conjugate was successfully synthesized the sample was sent for

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 efficiently since the major product (peaks at 9264.3 g/mol and 4064.2 g/mol) was too small.

• The theoretical mass of the the aptamer-DOTA conjugate is 9952.9 g/mol and the theoretical mass of the aptamer-DTPA conjugate is 9914.8 g/mol.

• This meant that the DTPA and DOTA chealators did not bind to the aptamer to form the aptamer-chelate conjugate.

• A small amount of the conjugate was synthesized successfully, however a side reaction with dmso resulted in an unstable final product.

• The peak at ~10030 g/mol in both spectra is evidence that the side reaction occurred.

• 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.


Conclution

• Amino-modified aptamer was successfully synthesized, purified and subsequently quantified in relatively high yield and purity compared to previous studies.

• Since time was not permiting future work will involve reacting the DNA once more with the DTPA/DOTA chelate to form the aptamer-chelate conjugate.

• Mass spectrometry will be used to confirm that the reaction was successful.

• The conjugates would then be tested in an MRI, in both buffer and serum, to find the best system for imaging thrombin.


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 DNA synthesizer, Gel electrophoresis setup, UV-Vis spectrometer, and Access to the 1.5 T MRI (Ottawa hospital) through a collaboration with Dr. Eve Tsai. I would also like to thank Erin McConnell and Maria DeRosa for their supervision. 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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