Abstract
Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.
Highlights
The significance of aptamers is increasingly appreciated by the scientific community and their diagnostic potential is attested by a vast number of publication describing the development of aptamer-based biosensors [1]
These assays rely on the so-called oligonucleotide-based enzyme capture assay (OECA), that is, the protein of interest selective aptamer is immobilized on the plate and the captured protein is detected through its enzyme activity by using fluorogenic substrates
Notwithstanding, the structural analysis of aptamer- and spiegelmer-protein complexes revealed that these oligonucleotides interact with their target through definite amino acid motifs; theoretically protein-selective spiegelmers can be generated without application of D-enantiomers of complete proteins [11,12]
Summary
The significance of aptamers is increasingly appreciated by the scientific community and their diagnostic potential is attested by a vast number of publication describing the development of aptamer-based biosensors [1]. Notwithstanding, the structural analysis of aptamer- and spiegelmer-protein complexes revealed that these oligonucleotides interact with their target through definite amino acid motifs; theoretically protein-selective spiegelmers can be generated without application of D-enantiomers of complete proteins [11,12]. This so-called domain approach of spiegelmer selection follows the rationality of antibody production, i.e., only a peptide motif of the protein of interest is used for triggering the immune response [13]. These spiegelmers were leveraged for developing an antibody-spiegelmer-composed homogenous sandwich assay that was suitable for selective detection of cTnI [15]
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