Rapid SERS Detection of Botulinum Neurotoxin Type A

Surface-enhanced Raman scattering (SERS) is a powerful technique for decoding of 2-5-component mixes of analytes. Low concentrations of analytes and complex biological media are usually non-decodable with SERS. Recognition molecules, such as antibodies and aptamers, provide an opportunity for a specific binding of ultra-low contents of analyte dissolved in complex biological media. Different approaches have been proposed to provide changes in SERS intensity of an external label upon binding of ultra-low contents of the analytes. In this paper, we propose a SERS-based sensor for the rapid and sensitive detection of botulinum toxin type A. The silver nanoisland SERS substrate was functionalized using an aptamer conjugated with a Raman label. The binding of the target affects the orientation of the label, providing changes in an analytical signal. This trick allowed detecting botulinum toxin type A in a one-stage manner without additional staining with a monotonous dose dependence and a limit of detection of 2.4 ng/mL. The proposed sensor architecture is consistent with the multiarray detection systems for multiplex analyses

Putative Mechanisms Underlying High Inhibitory Activities of Bimodular DNA Aptamers to Thrombin

Nucleic acid aptamers are prospective molecular recognizing elements. Similar to antibodies, aptamers are capable of providing specific recognition due to their spatial structure. However, the apparent simplicity of oligonucleotide folding is often elusive, as there is a balance between several conformations and, in some cases, oligomeric structures. This research is focused on establishing a thermodynamic background and the conformational heterogeneity of aptamers taking a series of thrombin DNA aptamers having G-quadruplex and duplex modules as an example. A series of aptamers with similar modular structures was characterized with spectroscopic and chromatographic techniques, providing examples of the conformational homogeneity of aptamers with high inhibitory activity, as well as a mixture of monomeric and oligomeric species for aptamers with low inhibitory activity. Thermodynamic parameters for aptamer unfolding were calculated, and their correlation with aptamer functional activity was found. Detailed analysis of thrombin complexes with G-quadruplex aptamers bound to exosite I revealed the similarity of the interfaces of aptamers with drastically different affinities to thrombin. It could be suggested that there are some events during complex formation that have a larger impact on the affinity than the states of initial and final macromolecules. Possible mechanisms of the complex formation and a role of the duplex module in the association process are discussed

Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA–DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals