Aptamers are single stranded DNA or RNA oligonucleotides that possess functional properties arising from their ability to bind to target molecules with high affinity. As they have a very high recognition specificity, they can effectively replace antibodies, minimizing undesired immune responses, and are therefore useful in a great range of domains, such as biosensing and therapeutics. Aptamers are also naturally found in the triggering process of riboswitches, where the DNA or RNA sequence is generated by SELEX-probing against the molecule of interest.
Our proof-of-concept experiments required the usage of a set of well-documented aptamers that would bind one specific molecule. We thus decided to use the following three thrombin-binding aptamers:
|Thrombin Aptamer 1||5’-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3’||Cy5 and Biotinylation|
|Thrombin Aptamer 2||5’-GGT TGG TGT GGT TGG-3’||Cy3|
|Thrombin Aptamer 3||5’-GAC ACA GGA CAT GAA ACT GAT GAG AAT AGA GCG AAA GGT TGGTGT GGT TGG-3’||Cy3|
Both of Thrombin Aptamers 1 and 2 were shown to recognize different epitopes of Human α-Thrombin. The Cy5-label of the first aptamer was removed, after proving that it specifically recognized thrombin, and replaced by a biotin extension at its 5’ end in order to perform a sandwich assay on a surface. The thrombin Aptamer 2 was later modified in order to include the short trigger of the Zika 27B toehold sequence at its 5’ end to become thrombin aptamer 3.
We later investigated another protein on which to perform a sandwich assay and expand our project: PDGF-BB, a dimeric form of PDGF (Platelet-Derived Growth Factor), which provides a direct application to diagnostics because of its key role in a number of pulmonary and fibrotic diseases. Again, two aptamers discovered by SELEX were used for the sandwich assay:
|PDGF Aptamer 1||5’-TAC TCA GGG CAC TGC AAG CAA TTG TGG TCC CAA TGG GCT CTG AGT A-3’||Biotinylation|
|PDGF Aptamer 2||5’- GAC ACA GGA CAT GAA ACT GAT GAG AAT AGA GCG AAA TAC TCA GGG CAC TGC AAG CAA TTG TGG TCC CAA TGG GCT CTG AGT A -3’||Cy3|
The second aptamer contains the trigger of the 27B toehold sequence at its 5’ end in order to induce the expression of a reporter gene.
The detection part of our biosensing device incorporates a sandwich assay. Inspired by the sandwich ELISA process, it consists of a biotinylated non-labeled aptamer that binds a streptavidin- or neutravidin-coated surface (binding with biotin provides one of the strongest non-covalent bonds found in nature).The aptamer will in turn detect the molecule of interest by binding to one of its epitopes on a target molecule, for example either thrombin or PDGF. A second aptamer will then recognize another epitope contained within the same target molecule. In our experiments, this second aptamer was first Cy3-labeled and then extended at its 5’ end to include the toehold trigger sequence, which we designed to induce the expression of a reporter protein.
Thrombin is a serine protease enzyme that plays a key role in blood clotting. During the process of blood clotting, the proteolytic cleavage of prothrombin (coagulation factor II) leads to the formation of thrombin. In our experiments, we are detecting Human α-Thrombin.
Many aptamers for this enzyme have been determined via Systematic Evolution of Ligands by EXponential enrichment (SELEX) and are now well documented we thus choose this protein to serve as the foundation for the proof-of-concept experiments for our project.
Platelet-Derived Growth Factor (PDGF)
PDGF is primarily a growth factor implicated in angiogenesis, the growth of blood vessels from pre-existing tissue. PDGF is a disulfide linked dimeric glycoprotein. It can either be a homozygous dimer of PDGF-A, -B, -C, and -D subunits or a heterodimeric protein (e.g. PDGF-AB). The PDGF signalling pathway, an autocrine pathway, is often linked to various diseases, most notably in epithelial cancer, where it affects tumor growth and metastasis, among other things.
Aptamers binding to this clinically important protein have been determined through SELEX and we decided to use said aptamers to further our proof-of-concept.
1. Tasset, D. M., Kubik, M. F., & Steiner, W. (1997). Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. Journal of molecular biology, 272(5), 688-698
2. Frederiksson S. et al. (2002). Protein detection using proximity-dependent DNA ligation assays. Nature Biotechnology 20, 473-477.
3. Lequin, R. M. (2005). Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clinical chemistry, 51(12), 2415-2418.