No abstract

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.

Background: Cell-free protein expression has become a widely used alternative of in vivo, cell-based systems in functional and structural studies of proteins. The wheat germ-based method outstands from the commercially available eukaryotic in vitro translation systems by its flexibility, high translation efficiency and success rate of properly folded eukaryotic protein synthesis. The original T7 promoter containing pEU3-NII vector was improved previously by addition of a ligation-independent cloning site, His 6-and GST-tags, and a TEV protease cleavage site to facilitate the creation of recombinant plasmids, permit affinity purification, and enable production of purified, tag-free target proteins, respectively. Results: Here, we describe a further development of pEU3-NII vector by inserting the rare-cutting, NotI restriction enzyme cleavage site to simplify vector linearization step prior to in vitro transcription. Additionally, His 12 , FLAG, and Halo affinity tag coding vectors have been created to increase detection sensitivity, specificity of interaction studies, and provide covalently linkable ligands for pull-down assays, respectively. Finally, the presented GST-His 6 , and GSTbiotin double-tagging vectors could broaden the range of possibilities of protein-protein interaction studies. Conclusions: The new generation of pEU3-NII vector family allows a more rapid production of translationally active mRNA and wheat germ cell-free expression of target proteins with a wide variety of affinity tags thus enables designing flexible and diverse experimental arrangement for in vitro studies of proteins.

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.

Background/Objective The RANK (Receptor Activator of Nuclear Factor kappa B) receptor, located on the surface of osteoclasts plays a critical role in osteoclastogenesis. Aptamers are single-stranded oligonucleotides selected from a random single stranded RNA/DNA library to bind specifically to various targets (e.g. proteins) after folding into well-defined spatial structures. We set out to develop aptamers targeting the RANK receptor, a key marker of osteoclast differentiation. Materials and methods A single-stranded DNA library (approx. 1012 different sequences) was amplified via polymerase chain reaction. Thiophosphate-modified deoxynucleotide triphosphates were used in order to generate nuclease-resistant thioaptamers. The glutathione S transferase-tagged, extracellular domain of the human RANK protein (AA 30–212) was produced by in vitro translation. After affinity purification, using this target, thioaptamers were selected by the iterative, in vitro SELEX (Systematic Evolution of Ligands by EXponential enrichment) process. The enriched single-stranded thioaptamer pool was amplified, cloned into bacteria, and 31 clones with inserts corresponding to the size of the aptamer were then sequenced. Thioaptamer candidates were screened by surface plasmon resonance (SPR). Results Thioaptamers were generated against the extracellular domain of the human RANK protein using the SELEX process. The interaction of nine randomly chosen thioaptamer candidates and their target protein was screened by SPR using the random initial oligonucleotide pool as negative control. A binding event was defined by a reflectivity variation (deltaR%) greater than 0,1. We identified four thioaptamers, which showed higher reflectivity variation (deltaR% 0.15–0.42), as opposed to the negative control (deltaR% 0.05), representing 1.4–3.9 fmol/mm2 and 0.5 fmol/mm2 surface coverage of the protein, respectively. Conclusion We identified four thioaptamers via the in vitro selection process, SELEX, which can recognise the extracellular domain of the human RANK protein. By further selection using transgenic cell lines and murine osteoclast cultures our aim is to select aptamers, which can specifically bind to both the murine and human RANK protein. Such nuclease-resistant aptamers may facilitate the identification of osteoclast precursors in human serum and may also be of potential therapeutic value as neutralising agents against RANK in murine models of inflammatory bone loss.