Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

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.

A simple modification increases specificity and efficiency of asymmetric PCR.

Although various methods have been developed to suffice the oligonucleotide demand of molecular biology laboratories, in vitro production of high-purity ssDNAs remains to be a challenging task. We hypothesized that complementing the asymmetric PCR with 3' phosphate blocked limiting primer decreases the mispriming thus reduces polymerisation of DNA by-products. The presented results attest our assumption that the primer blocked asymmetric PCR (PBA-PCR) selectively produces ssDNA of interest and is even suitable for effective amplification of DNA libraries of large sequence space. The high-throughput sequence analysis demonstrated that PBA-PCR also alleviates the PCR bias obstacle since it does not distort the sequence space. The practicability of the novel method was verified by monitoring the process of SELEX and screening of aptamer candidates using PBA-PCR produced ssDNAs in Amplified Luminescent Proximity Homogeneous Assay. In summary, we have developed a generally applicable method for straightforward, cost-effective production of ssDNA with on demand labelling.

Aptamers for respiratory syncytial virus detection.

The identification of the infectious agents is pivotal for appropriate care of patients with viral diseases. Current viral diagnostics rely on selective detection of viral nucleic acid or protein components. In general, detection of proteins rather than nucleic acids is technically more suitable for rapid tests. However, protein-based virus identification methods depend on antibodies limiting the practical applicability of these approaches. Aptamers rival antibodies in target selectivity and binding affinity, and excel in terms of robustness and cost of synthesis. Although aptamers have been generated for virus identification in laboratory settings, their introduction into routine virus diagnostics has not been realized, yet. Here, we demonstrate that the rationally designed SELEX protocol can be applied on whole virus to select aptamers, which can potentially be applied for viral diagnostics. This approach does not require purified virus protein or complicated virus purification. The presented data also illustrate that corroborating the functionality of aptamers with various approaches is essential to pinpoint the most appropriate aptamer amongst the panel of candidates obtained by the selection. Our protocol yielded aptamers capable of detecting respiratory syncytial virus (RSV), an important pathogen causing severe disease especially in young infants, at clinically relevant concentrations in complex matrices.

Is less more? Lessons from aptamer selection strategies.

Aptamers have many inherent advantages originating from their in vitro selection and tailored chemical synthesis that makes them appealing alternatives of antibodies in bioaffinity assays. However, what ultimately matters, and that is the prerequisite to give way to all these advantages, is how well, and how selectively the aptamers bind to their targets. With the aptamer selection largely in the hand of life scientists, analytical chemists focused mostly on methodological development of aptamer-based assays using a fairly restricted number of aptamers to prove their concepts. However, ideally the development of an aptamer-based assay should start from the selection of aptamers to ensure their proper functionality in real samples. For instance information on the sample matrix can be implemented within counter-selection steps to discard aptamer candidates that show cross-reactivity to matrix components or critical interferents. In general, a larger consideration of the analytical use during selection and characterization of aptamers have been shown to increase the applicability of aptamers. Therefore, this review is a short, subjective view on trends in aptamer development highlighting factors to consider during their selection for a successful analytical application.

A rational approach for generating cardiac troponin I selective Spiegelmers.

We report the first protein selective Spiegelmers of diagnostic relevance by rational identification of a target epitope and reverse screening of Spiegelmer candidates following the selection procedure. Application of the presented approach resulted in isolation of cardiac troponin I selective Spiegelmers with low nanomolar dissociation constant and functionality in serum.