To cite this article: Protopopova AD, Barinov NA, Zavyalova EG, Kopylov AM, Sergienko VI, Klinov DV. Visualization of fibrinogen aC regions and their arrangement during fibrin network formation by high-resolution AFM. J Thromb Haemost 2015; 13: 570-9.See also Rocco M, Weisel JW. Exposed: the elusive aC regions in fibrinogen, fibrin protofibrils and fibers. This issue, pp 567-9.Summary. Background: Fibrinogen has been intensively studied with transmission electron microscopy and x-ray diffraction. But until now, a complete 3D structure of the molecule has not yet been available because the two highly flexible aC regions could not be resolved in fibrinogen crystals. This study was aimed at determining whether the aC regions can be visualized by high-resolution atomic force microscopy. Methods: Atomic force microscopy with super high resolution was used to image single molecules of fibrinogen and fibrin associates. The key approach was to use a graphite surface modified with the monolayer of amphiphilic carbohydrate-glycine molecules and unique supersharp cantilevers with 1 nm tip diameter. Results: Fibrinogen aC regions were visualized along with the complete domain structure of the protein.In almost all molecules at pH 7.4 the D domain regions had one or two protrusions of average height 0.4 AE 0.1 nm and length 21 AE 6 nm. The complex, formed between thrombin and fibrinogen, was also visualized. Images of growing fibrin fibers with clearly visible aC regions have been obtained. Conclusions: Fibrin aC regions were visible in protofibrils and large fibers; aC regions intertwined near a branchpoint and looked like a zipper. These results support the idea that aC regions are involved in the thickening of fibrin fibers. In addition, new details were revealed about the behavior of individual fibrin molecules during formation of the fibrin network. Under the diluted condition, the positioning of the aC regions could suggest their involvement in long-range interactions between fibrin but not fibrinogen molecules.

Highly sensitive and rapid technology of surface enhanced Raman scattering (SERS) was applied to create aptasensors for influenza virus detection. SERS achieves 10 6 −10 9 times signal amplification, yielding excellent sensitivity, whereas aptamers to hemagglutinin provide a specific recognition of the influenza virus. Aptamer RHA0385 was demonstrated to have essentially broad strain-specificity toward both recombinant hemagglutinins and the whole viruses. To achieve high sensitivity, a sandwich of primary aptamers, influenza virus and secondary aptamers was assembled. Primary aptamers were attached to metal particles of a SERS substrate, and influenza viruses were captured and bound with secondary aptamers labelled with Raman-active molecules. The signal was affected by the concentration of both primary and secondary aptamers. The limit of detection was as low as 1 · 10 −4 hemagglutination units per probe as tested for the H3N2 virus (A/England/42/72). Aptamer-based sensors provided recognition of various influenza viral strains, including H1, H3, and H5 hemagglutinin subtypes. Therefore, the aptasensors could be applied for fast and low-cost strain-independent determination of influenza viruses.

During the COVID-19 pandemic, the development of sensitive and rapid techniques for detection of viruses have become vital. Surface-enhanced Raman scattering (SERS) is an appropriate tool for new techniques due to its high sensitivity. SERS materials modified with short-structured oligonucleotides (DNA aptamers) provide specificity for SERS biosensors. Existing SERS-based aptasensors for rapid virus detection are either inapplicable for quantitative determination or have sophisticated and expensive construction and implementation. In this paper, we provide a SERS-aptasensor based on colloidal solutions which combines rapidity and specificity in quantitative determination of SARS-CoV-2 virus, discriminating it from the other respiratory viruses.

Viral infections are among the main causes of morbidity and mortality of humans; sensitive and specific diagnostic methods for the rapid identification of viral pathogens are required. Surface-enhanced Raman spectroscopy (SERS) is one of the most promising techniques for routine analysis due to its excellent sensitivity, simple and low-cost instrumentation and minimal required sample preparation. The outstanding sensitivity of SERS is achieved due to tiny nanostructures which must be assembled before or during the analysis. As for specificity, it may be provided using recognition elements. Antibodies, complimentary nucleic acids and aptamers are the most usable recognition elements for virus identification. Here, SERS-based biosensors for virus identification with oligonucleotides as recognition elements are reviewed, and the potential of these biosensors is discussed.

Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K, Na, NH, Ba, and Sr; on the contrary, Mn was coordinated in the grooves, outside the G-quadruplex. K or Na coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K coordination provided the well-known high inhibitory activity of the aptamer, whereas Na coordination supported low activity. Although NH coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba and Sr coordination. Mn coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a different sequence of loops in the G-quadruplex module provided an equilibrium of antiparallel and parallel G-quadruplexes that shifted with cation binding. In conclusion, structures of G-quadruplex aptamers are flexible enough and are fine-tuned with different cation coordination.