Post on 12-Nov-2014
description
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
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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 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.
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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.
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R1 = the chelator (DTPA and DOTA)R2= the aptamer
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Results
Figure 3: PAGE purification. Left : Aptamer-DOTA conjugate. Right : Aptamer-DTPA conjugate.
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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.
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Results
Figure 4: UV-vis absorbance spectrum of DNA for quantification of the DNA-chelate conjugates.
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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 the Beer-Lambert 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.
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Results
Daphnée Dubouchet-Olsheski
Figure 5: Calibration curve from Xylenol Orange Test.
Figure 6: Eppendorf tubes containing Xylenol Orange Test reactions.
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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 flow through (FT) from the Gd(III) incubation was compared with known concentrations of Gd(III) as represented in Figures 6 and 7.
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Results
Figure 7: UV-vis absorbance spectrum of Xylenol Orange Test calibration curve.
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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.
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Results
Figure 8: Mass spectrometry results
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Results
Figure 8: Mass spectrometry results
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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.
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Conclusion
• Amino-modified aptamer was successfully synthesized, purified and, subsequently, quantified in relatively high yield and purity compared to previous studies.
• Since time was not permitting, 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.
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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 graciously 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. 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.