DNA Aptamers That Bind to MUC1 Tumour Marker: Design and Characterization of MUC1-Binding Single-Stranded DNA Aptamers

Abstract

Agents able to bind tightly and selectively to disease markers can greatly benefit disease diagnosis and therapy. Aptamers are functional molecules, usually DNA or RNA oligonucleotides, with the appropriate sequence and structure to form a complex with a target molecule. MUC1 is a well-known tumour marker present in a variety of malignant tumours and it has been a target of interest for many years. In this work we report the selection of DNA aptamers that bind with high affinity and selectivity to the MUC1 peptides. Combinatorial chemistry techniques based on the SELEX methodology were used for the identification of the specific aptamers. These were selected from an initial library containing a 25-base-long variable region, resulting in 4²⁵ random sequences of single-stranded DNA molecules, for their ability to bind to synthetic forms of MUC1. Ten rounds of in vitro selection were performed enriching for MUC1 binding. By round ten more than 90% of the pool of sequences consisted of MUC1-binding molecules. Selected aptamer families were cloned, sequenced and found to be unique, sharing no sequence consensus. The binding properties of these aptamers were quantitated by enzyme-linked immunosorbent assay and surface plasmon resonance, whereas their specificity for MUC1-expressing cancer cells has been validated using fluorescent microscopy. Aptamers offer significant advantages over existing antibody-based recognition procedures in that they offer higher binding affinity (higher retention/reduced dissociation) and specificity to the target (ability to determine variations on the protein target down to single amino acid changes), higher selectivity against mutated protein epitopes and potentially reduced immunogenicity and increased tumour penetration associated with their size.

Clinical applications of nucleic acid aptamers in cancer

“…In addition, Muc1 is an attractive target, as its overexpression has been associated with various types of cancer and its distribution pattern changes on the cell surface (82,83). DNA aptamers directed against Muc1 (AptA and AptB) were identified in 2006 (84) and have since been implemented in ex vivo imaging and radiotherapy (85). In subsequent studies, when such an DNA aptamer was coupled to the light-activated photodynamic therapy agent chlorine e6, it specifically delivered chlorine e6 into epithelial cancer cells, with a significant enhancement in efficiency (>500-fold increase) compared to that of the drug alone, and achieved cancer-specific cytotoxicity (86).…”

Targeted Nanoparticles for Cancer Therapy

“…A large number of aptamers have been raised against cancer-associated antigens such us AS1411 aptamer for targeting nucleolin protein, which is highly expressed in the membrane of cancer cells [48][49], aptamers CPG 7909 and IMO 2055, that target Toll-like receptor 9 (TLR9), which is expressed by certain immune cells, TD05 aptamer, which was selected for the Burkitt's lymphoma Ramos cell line [47], Sgc8c aptamer which targets leukemia biomarker protein tyrosine kinase-7 (PTK7) [46,50] and can recognize target leukemia cells, DNA aptamers to leukemic lymphoid (CEM) cells [46], and fruoropyrimidine RNA aptamers which target Prostate-specific membrane antigen (PSMA) [51] for targeting prostate cancer. And also another aptamers against antigens such as pigpen [52] for targeting the tumor microvasculature, or mucin 1 (MUC1) [53] for targeting various epithelial neoplasms that upregulate MUC1, whose expression has been associated with carcinomas.…”

Anti-MUC1 aptamers: radiolabelling with 99mTc and biodistribution in MCF-7 tumour-bearing mice

“…AptA and MAG2-AptA, dissolved in the same solution previously used for the selection [22], are treated with the Tc-tartrate, having R t of 23.7 and 2.7 min respectively, were 7 detected. The radiolabelled aptamers were isolated from small molecular weight impurities by ultrafiltration using microcon 3kDa MWCO filters with a recovery of >80% of the radioactivity on the cartridge.…”

“…The selection of high affinity and specificity DNA aptamers against the highly immunogenic epitope of the un-glycosilated protein core of the MUC1 glycoprotein [22] and for the malignant glycosilated form of MUC1 [23] as tumour markers on epithelial cancer cells has been previously reported.…”