mucins and specifically the MUC1 are such great tum
or markers for epithelial malignancies, they are more readily being used for immunotherapeutic and diagnostic approaches (2). Using aptamer binding techniques to target the specific MUC1 glycoprotein would allow for the formation of new diagnostic assays against the tumor to help with early diagnosis of several types of epithelial malignancies (4).
Figure 1: MUC1 structure
Aptamers are nucleic acid oligonucleotides that bind with specificity and tightness to areas of interest. Developing aptamers against certain targets makes it possible to inhibit the target’s f
unction, consequently reducing the effects of a disease they are linked to (5). Other research has shown that using the SELEX methodology to choose aptamers against MUC1 provided insight to the exact binding site between the aptamer and MUC1 complexes which gives hope to the possibility to improving this detection methodology for further use in early diagnosis techniques (6).
Specific Aim 1:
MUC1 is an excellent epithelial tumor antigen in several different carcinomas around the body. It has been shown that by binding aptamers to MUC1, isolating cancer cells at early stag
es of malignancies can be greatly beneficial to the populace. Thus the selection of RNA aptamers against MUC1 will make cancer cells more apparent at an early stage and destroying those specific cancer cells will become easier.
Figure 2: Mucin acts as an antigen on the surface of most epithelial cancer cells
GenScript USA $54.00 1mg Catalog Number RP20402_1mg
References:
- Ho, J.J.L. "Mucins in the Diagnosis and Therapy of Pancreatic Cancer." Current Pharmaceutical Design 6.18 (2000): 1881-896. Print.
- Parry, S. "Identification of MUC1 Proteolytic Cleavage Sites in Vivo." Biochemical and Biophysical Research Communications 283.3 (2001): 715-20. Print.
- Cheng, Alan K. H., Huaipeng Su, Y. Andrew Wang, and Hua-Zhong Yu. "Aptamer-Based Detection of Epithelial Tumor Marker Mucin 1 with Quantum Dot-Based Fluorescence Readout." Analytical Chemistry 81.15 (2009): 6130-139. Print.
- Ferreira CS, and Papamichael K. "DNA Aptamers against the MUC1 Tumour Marker: Design of Aptamer-antibody Sandwich ELISA for the Early Diagnosis of Epithelial Tumours." Anal Bioanal Chem (2007): 1039-050. Print.
- Cerchia L, and De Franciscis V. "Targeting Cancer Cells with Nucleic Acid Aptamers." Trends Biotechnol. (2010). Print.
- Cheng AK,, Su H, Wang YA, and Yu HZ. "Aptamer-based Detection of Epithelial Tumor Marker Mucin 1 with Quantum Dot-based Fluorescence Readout." Anal Chem (2009): 6130-139. Print.
9 comments:
Good work.
How will your selection differ from Ferreira et al's selection? How will you improve upon the aptamer they've selected? Did they select a DNA aptamer & will you select an RNA aptamer?
As you've pointed out, it will be important for your target to be glycosolated (can't spell). Will you purchase human Muc1 with the correct glycosolation? Do you anticipate any target issues?
How is this aptamer better than other methods of diagnosing? The description of aptamers seems too much like something from a source, maybe make it more your own!
In the paper for the aptamer, it appears to be a DNA aptamer. UT doesn't have access to the original paper published in Tumor Biology (Ferreira, C. S. M., Matthews, C. S. and Missailidis, S. Tumor Biol. 2006, 27, 289–301), but I have contacted the authors for a reprint.
In the QD paper (your ref #6), the target MUC-1 was a custom synthesis: "The MUC1 peptide with two repeats of the 20 amino acid variable tandem repeat region (from the N terminus to the C terminus: PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA) was purchased as a custom synthetic peptide from the University of British Columbia Brain Research Centre (Vancouver, BC)." The 20 amino acid variable region is extracellular and potentially can be detected in the blood stream. Abcam makes the MUC1 HIS tagged, Cat# ab80082 but it may not be the extracellular immunogenic portion. AnaSpec also makes a 9aa peptide with the sequence H - Ser - Thr - Ala - Pro - Pro - Ala - His - Gly - Val - OH, Cat# 61357. This is a portion of the sequence used above, but we would need to functionalize onto a biotin and somehow remove the remaining free biotin. It won't be an easy target, but an RNA aptamer may be particularly interesting for diagnostics, especially if it has better binding kinetics than the original DNA aptamer.
It seems that the C-terminal region is located in the cytoplasm per Gendler and Spicer's 1995 work. It would be interesting to select a diagnostic aptamer for the cytoplasmic portion given that the protein is often found in the blood stream when over expressed by epithelial cancer cells suggested by Cheng et al. I also found the sequence from GenBank that matched the AbCam sequence. GenBank suggests that portion could be cytoplasmic. So lets just order it.
That original paper on MUC-1 can be found here.
The Abcam company got back to me with a price. This product is 250ug for $255.00 and in stock at the moment. The ID number is ab80082 which is a MUC1 protein (His tag)
http://www.abcam.com/index.html?datasheet=80082).
MUC-1 came in a few days ago (9-15-10?). It was in PBS solution at 250ug/250ul. The target is 10kDa so it is 100uM concentration. Sabeena aliquoted a few different volumes (pmol/tube) @ 800, 400 and 200. Lot# 897283. Aliquoted and placed in -80C rack J3 second box back.
custom peptide synthesis- The information that you provided was helpful for readers. I will have to share your article with others.
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