Slide Presentation

  • 81 views
Uploaded on

 

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads

Views

Total Views
81
On Slideshare
0
From Embeds
0
Number of Embeds
0

Actions

Shares
Downloads
1
Comments
0
Likes
0

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide
  • 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.
  • Thrombinis an enzyme in blood plasma that causes the clotting of blood by converting fibrinogen to fibrin.Anticoagulants (a protein that inhibits clotting) are always present in the blood stream. When a blood vessel tears the amount of procoagulants (a prontein that stimulates clotting) increase. And coagulation (the clotting of blood) begins. However when the amount of anticoagulants and procoagulants is not perfectly balanced complication arise. Many factors can contribute to venous blood clotting such as obesity, prolonged immobility and age (over 60). Blood clots can be very dangerous as they can break loose and move to other parts of your body. A blood clot in lungs is called a pulmonary embolism.A blood clot in the brain can block the flow of oxygen to the brain and cause a stroke. MR imaging of thrombin could be useful in the imaging and isolation of blood clots (thrombus and embolus). *embolus is any detached, traveling intravascular carried by circulation.The targeting of thrombin could prove useful for precise MR imaging of internal bleeding and blood clotting processes. A 29 base long DNA aptamerthat 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 is short for Magnetic Resonance Imaging. It 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). Relaxation time is the time it takes for the protons to get back to their original alignment after they have been hit by the radio wave. The relaxation time depends on the environment.That's why different tissues have different contrasts on an MRI image.Ex. Lighter spots are denser than darker spots. 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 tocreate specific receptor molecules (Aptamers) that can target specific tissues such as thrombin.
  • MRI is short for Magnetic Resonance Imaging. It 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). Relaxation time is the time it takes for the protons to get back to their original alignment after they have been hit by the radio wave. The relaxation time depends on the environment.That's why different tissues have different contrasts on an MRI image.Ex. Lighter spots are denser than darker spots. 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 tocreate specific receptor molecules (Aptamers) that can target specific tissues such as thrombin.
  • 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.
  • R1 = the chelator (DTPA and DOTA)R2= the aptamerThepreparation of the conjugate included - Synthesizing the DNA using the MerMade SoftwareReacting the DNA columns with the chelate (DTPA or DOTA)Recoveringolingonucleotide from gelDesalting the DNAPass the conjugate through a UV visto make sure the aptamer and chelate are together.
  • 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.
  • 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.
  • 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.
  • 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) 
  • PicutresDNA https://team.inria.fr/zenith/files/2013/01/health-dna-backgrounds-powerpoint.gifBlood Clothttp://www.knowabouthealth.com/wp-content/uploads/2011/10/blood_clotting-4.jpgMRI http://www.turbosquid.com/3d-models/mri-medical-imaging-machine-3d-model/373001Gadoliniumhttp://www.acceleratingfuture.com/michael/blog/2010/01/our-friend-gadolinium/Contrast Agenthttp://www.nzbri.org/research/labs/mri.phpResearch(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 thelanthanoids and xylenol orange” Microchim. Acta 1968, 56, 1059-1065.

Transcript

  • 1. Development Of An Aptamer-Based MRI Contrast Agent For Thrombin Detection Daphnée Dubouchet-Olsheski under the supervision of Erin McConnell, Maria DeRosa. March something07/03/2013 Daphnée Dubouchet-Olsheski 1
  • 2. Outline • Background Information • Aptamers • Thrombin • MRI • Introduction • Project Goals • Preparation of the Conjugate • Results and Interpretations • Relevant Application • References and Acknowledgements07/03/2013 Daphnée Dubouchet-Olsheski 2
  • 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.07/03/2013 Daphnée Dubouchet-Olsheski 3
  • 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.07/03/2013 Daphnée Dubouchet-Olsheski 4
  • 5. MRI D A B Magnetic Resonance Imaging Machine Gadolinium SCN-DTPA E C F Contrast Agent SCN-DOTA07/03/2013 Daphnée Dubouchet-Olsheski 5
  • 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.07/03/2013 Daphnée Dubouchet-Olsheski 6
  • 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.07/03/2013 Daphnée Dubouchet-Olsheski 7
  • 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.07/03/2013 Daphnée Dubouchet-Olsheski 8
  • 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.07/03/2013 Daphnée Dubouchet-Olsheski 9
  • 10. Results The yield and purity of the amino-modified aptamers was low.07/03/2013 Daphnée Dubouchet-Olsheski 10
  • 11. Results07/03/2013 Daphnée Dubouchet-Olsheski 11
  • 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.07/03/2013 Daphnée Dubouchet-Olsheski 12
  • 13. Results07/03/2013 Daphnée Dubouchet-Olsheski 13
  • 14. Results07/03/2013 Daphnée Dubouchet-Olsheski 14
  • 15. Results07/03/2013 Daphnée Dubouchet-Olsheski 15
  • 16. Results07/03/2013 Daphnée Dubouchet-Olsheski 16
  • 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)07/03/2013 Daphnée Dubouchet-Olsheski 17
  • 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, 1059-1065.07/03/2013 Daphnée Dubouchet-Olsheski 18