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.

The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells.

Aptamers are functional molecules able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. MUC1 is a well-known tumour marker present in epithelial malignancies and is used in immunotherapeutic and diagnostic approaches. We report the selection of DNA aptamers that bind with high affinity and selectivity an MUC1 recombinant protein containing five repeats of the variable tandem repeat region. Aptamers were selected using the SELEX methodology from an initial library containing a 25-base-long variable region for their ability to bind to the unglycosylated form of the MUC1 protein. After ten rounds of in vitro selection and amplification, more than 90% of the pool of sequences consisted of target-binding molecules, which were cloned, sequenced and found to share no sequence consensus. The binding properties of these aptamers were quantified using ELISA and surface plasmon resonance. The lead aptamer sequence was subsequently used in the design of an aptamer-antibody hybrid sandwich ELISA for the identification and quantification of MUC1 in buffered solutions. Following optimisation of the operating conditions, the resulting enzyme immunoassay displayed an EC50 value of 25 microg/ml, a detection limit of 1 microg/ml and a linear range between 8 and 100 microg/ml for the MUC1 five tandem repeat analyte. In addition, recovery studies performed in buffer conditions resulted in averaged recoveries between 98.2 and 101.7% for all spiked samples, demonstrating the usability of the aptamer as a receptor in microtitre-based assays. Our results aim towards the formation of new diagnostic assays against this tumour marker for the early diagnosis of primary or metastatic disease in breast, bladder and other epithelial tumours.