Disorders of the blood coagulation cascade continue to pose a major clinical challenge, necessitating the development of new therapeutic agents capable of modulating this process. Several oligonucleotide aptamers targeting coagulation factors have been developed, and some are undergoing preclinical or clinical evaluation. Among them, anti-thrombin anticoagulant aptamers are promising dual-targeting agents in that, in addition to inhibiting enzyme activity, they may limit thrombin generation by binding to its precursor, prothrombin. In the present study, combined calorimetric and spectroscopic analyses reveal that these aptamers recognize proexosite I of prothrombin and exosite I of thrombin through broadly similar thermodynamic binding mechanisms. Integration of structural small-angle X-ray scattering (SAXS) studies and limited proteolysis assays shows that aptamer binding to proexosite I alters prothrombin structure, shifting the equilibrium from its more abundant closed form to the open conformation. Taken together, these results support the classification of these aptamers as dual-targeting agents capable of recognizing both thrombin and prothrombin and provide guidance for their continued development as anticoagulant therapeutics.

Disorders of the blood coagulation remain a leading cause of death and disability worldwide raising the search for therapeutic agents able to modulate the coagulation cascade. Different oligonucleotide aptamers have been selected against different coagulation factors and some of them are in preclinical or clinical studies. In particular, anti-thrombin aptamers are promising drugs as they inhibit the activity of the α-thrombin and, simultaneously, limit thrombin production via prothrombinase by binding its precursor prothrombin. To investigate the interaction of these aptamers with prothrombin, we performed extensive analyses using calorimetric and spectroscopic techniques, which suggested that they recognize proexosite I of prothrombin and exosite I of thrombin with comparable affinity. SAXS experiments performed on the complex formed by the protein and NU172, the only anti-thrombin aptamer in advanced clinical trials, provided structural insights into aptamer-prothrombin recognition. Interestingly, the aptamer binding to proexosite I shifts the open-closed equilibrium of prothrombin toward the open conformation. A reasonable mechanism underlying the effects of anti-thrombin aptamers towards prothrombin conversion into thrombin has been proposed. Altogether, these results definitively qualify these aptamers as bitargeted drugs, being able to modulate both thrombin function and generation, and supply structural bases to design new anticoagulants, which lack health side effects. ### Competing Interest Statement The authors have declared no competing interest.

The process of unfolding of G-quadruplex structure in the RE31 DNA-aptamer and in its complex with thrombin under the action of the fluorescently labeled complementary oligonucleotides of varying length with formation of double-helix structures has been studied. It has been suggested that G-quadruplex unfolding involves formation of an intermediate complex with an oligonucleotide. Thermodynamic parameters and kinetics of unfolding of the free aptamer and its complex with thrombin differ. Extension of the oligonucleotide sequence complementary to G-quadruplex by two nucleotides to cover the so-called “hinge region” had little impact on the conformational transition of G-quadruplex of the free aptamer. However, a pronounced effect has been observed for the aptamer–protein complex. Most likely these differences could be explained by the thrombin-induced conformational transition of the aptamer involving the hinge region.

DNA aptamers are oligonucleotides specifically bound to target molecules that can serve as antibodies of nucleic acid nature. For diagnosing the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), methods using antibodies specific to antigens on the virus are broadly used. We generated by classical SELEX a number of aptamers, interacting with the receptor-binding domain of SARS-CoV2 spike protein (SARS-CoV2 Spike RBD) from Wuhan-Hu-1 strain. The sequence identification was performed using a novel methodology based on the nanopore sequencing. For sequence identification of selected aptamers, we created the novel protocol for aptamer identification based on nanopore sequencing. We identified the best aptamer candidate named MEZ. It was chemically synthesized and tested for binding with SARS CoV2 Spike RBD domain of the S-protein from different strains. Kd of the complex is 6.5 nM being comparable with known aptamers. Virus neutralization tests demonstrate similar results for already known and MEZ aptamers. We identified differences for aptamers binding to SARS-CoV-2 Spike RBD from Wuhan-Hu-1 and Omicron strains. MD simulations reveal that the number of hydrogen bonds between the protein and aptamer is higher for the more stable complex. Moreover, dynamic network analysis show that the motions of the aptamer and protein are correlated to a higher extent in a more stable complex. Based on the experimental data and computational results we can conclude that the authentic RBD-aptamer complex has two specific points for interaction and the 3'-end of aptamer is responsible for strain identification. Therefore, the selected aptamer based on experimental data can be an alternative biological element for the development of SARS-CoV-2 diagnostic testing with strain specificity and cost efficiency due to the short length of aptamer being 31 nucleotides.

The aim of this study was to examine 3D structures of DNA aptamers, thrombin inhibitors. •The main objective was to study 3D structure 15TBA, RE31, NU172 aptamers using the small-angle X-ray scattering method. The size of 15TBA was 4.5 nm, which corresponds to a partially unfolded conformation. The CD spectrum of Nu172 in the presence of 50 mM strontium ions indicates the presence of an antiparallel G-quadruplex, the concentration o f which drops at 50°C. NU172 does not have a rigid structure, apparently due to the presence of a guanine residue in the GT loop. The NU172 aptamer does not form a stable conformation in solution either without ions or with Ba2+ and Sr2+ ions. • It was shown that there is possibility of aptamers transition from one conformation to another dependently on concentration and temperature confirms that the potassium ion is a unique stabilizing ion of natural molecules containing G-quadruplexes

The high potential of aptamers – specifi c molecular agents based on short single-stranded nucleic acids – makes high demands on the molecules under development for the effi ciency of interaction with target biomolecules. In this work, approaches are considered for studying the spatial structure of DNA aptamers in solution using various complementary methods, which make it possible to obtain a more complete picture of the formation of the structure and conformational changes, to track the interaction with the target protein, the tendency to oligomerization, and to characterize the spatial structure of both individual molecules and complexes.

The selective properties of a solution of oligonucleotide specific to IL-6 on the concentration of IL-6 in mixed saliva of patients with oral inflammatory processes were studied using SDS-PAGE by electrophoresis and enzyme immunoassay. The application of these methods showed that in the mixed saliva of patients after rinsing with a solution of an oligonucleotide specific for IL-6, the amount of IL-6 decreases. The ELISA Kit and 20% SDS-PAGE showed the highest sensitivity to determine the concentration of IL-6 in saliva, which should be considered in clinical laboratory practice.

Nucleic acid (NA) aptamers bind to their targets with high affinity and selectivity. The three-dimensional (3D) structures of aptamers play a major role in these non-covalent interactions. Here, we use a four-step approach to determine a true 3D structure of aptamers in solution using small-angle X-ray scattering (SAXS) and molecular structure restoration (MSR). The approach consists of (i) acquiring SAXS experimental data of an aptamer in solution, (ii) building a spatial distribution of the molecule’s electron density using SAXS results, (iii) constructing a 3D model of the aptamer from its nucleotide primary sequence and secondary structure, and (iv) comparing and refining the modeled 3D structures with the experimental SAXS model. In the proof-of-principle we analyzed the 3D structure of RE31 aptamer to thrombin in a native free state at different temperatures and validated it by circular dichroism (CD). The resulting 3D structure of RE31 has the most energetically favorable conformation and the same elements such as a B-form duplex, non-complementary region, and two G-quartets which were previously reported by X-ray diffraction (XRD) from a single crystal. More broadly, this study demonstrates the complementary approach for constructing and adjusting the 3D structures of aptamers, DNAzymes, and ribozymes in solution, and could supply new opportunities for developing functional nucleic acids.

DNA aptamers (oligonucleotides) interacting with thrombin exosite I contain G-quadruplex, two T-T, and one T-G-T loops in their structure. They prevent exosite I binding with fibrinogen and thrombin receptors on platelet surface, thereby suppressing thrombin-stimulated formation of fibrin from fibrinogen and platelet aggregation. Earlier, we synthe-sized original antithrombin aptamer RE31 (5′-GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC-3′) that contained (in addition to G-quadruplex) a hinge region connected to six pairs of complementary bases (duplex region). In this study, we compared properties of RE31 aptamer and its analogues containing varying number of bases in the duplex region and nucleotide insertions in the hinge region. Reduction in the number of nucleotides in the duplex region by 1 to 4 pairs (in comparison with RE31 aptamer) resulted in the decrease of the structural stability of aptamers (manifested as lower melting temperatures) and their ability to inhibit thrombin-stimulated fibrin formation in human blood plasma in tests of thrombin, prothrombin, and activated partial thromboplastin times. However, an increase in the number of bases by 1 to 2 pairs did not cause significant changes in the stability and antithrombin activity of the aptamers. Insertions into the hinge region of RE31 aptamer decreased its antithrombin activity. Investigation of RE31 antithrombotic properties demonstrated that RE31 (i) slowed down thrombin formation in human blood plasma (thrombin generation test), (ii) accelerated lysis of fibrin clot by tissue plasminogen activator in in vitro model, and (iii) suppressed arterial thrombosis in in vivo model. Based on the obtained data, RE31 aptamer can be considered as a potentially effective antithrombotic compound.

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain.

Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer. The crystal structure of thrombin-NU172 complex reveals a bimodular duplex/quadruplex architecture for the aptamer, which binds thrombin exosite I through a highly complementary surface involving all three loops of the G-quadruplex module. Although the duplex domain does not interact directly with thrombin, the features of the duplex/quadruplex junction and the solution data on two newly designed NU172 mutants indicate that the duplex moiety is important for the optimization of the protein-ligand interaction and for the inhibition of the enzyme activity. Our work discloses the structural features determining the inhibition of thrombin by NU172 and put the basis for the design of mutants with improved properties.

Experiments with white rats were carried out to study the effects of original antithrombin DNA aptamers on the renal function and animal survival in rhabdomyolisis caused by intramuscular injection of a hyperosmolar glycerol solution. The DNA aptamers were demonstrated to exert a nephroprotective effect, reducing a retention azotemia and proteinuria, and improving the renal excretory function and overall survival of animals.

Antithrombin DNA aptamersRE31 are single-chain oligonucleotides that fold into three-dimensional forms allowing them to bind the enzyme with high affinity and inhibit its activity in vivo. They are rapidly degraded by a nonspecific nuclease, and, to prolong the lifetime of the aptamer DNA in the bloodstream, it is necessary to coat it with a polymer envelope. A new approach to solving this problem based on preparation of DNA-polyelectrolyte complexes with a minimal particle size that can circulate with blood flow. In our experiments, the negatively charged aptamer DNA RE31 was coated step-by-step with positively charged protamine. They had protamine/aptamer ratios of 0.2/1 and 0.4/1 by charge, with particle size being determined by dynamic light scattering. The aptamer DNA-protamine complexes were administered to rats, followed by ex vivo analysis of blood samples. The results showed that prothrombin time (PT) increased by a factor of 5.6-6.7 within 2 h after injection and remained at approximately the same level for 6 h, while injections of pure protamine did not lead to any noticeable change in clotting time. Thus, complexation with protamine proved to prolong the inhibitory activity of the RE31 DNA aptamer.

The binding to Lon protease through biotinylated aptamers whose structures contain G-quadruplex fragments with magnetic nanoparticles (MNPs) functionalized by streptavidin was investigated. The conditions of binding of target aptamers to MNPs are met. The resulting complexes are proposed for detection of Lon protease in different biological sources and for constructing a novel biomagnetic nanosensor immunoassay system.

A ptamers are a new class of oligonucleotide compounds capable of specific binding to various molecular targets and inhibiting their activity. Aptamers are selected from a library of randomly syn-thesized oligonucleotides (from 20 to 60 nucleotides long) based on their ability to bind to the target molecule. In the future, such primary aptamers can be chemically modified to optimize their structure and increase stability. Aptamers are considered to be chemical (oligonucleotide) analogues of monoclonal antibodies: their specificity is similar to that of antibodies, and they have high affinity to their targets. Aptamers are widely used to create pharmacological medicines. As pharmacological substances, they have a number of benefits over antibodies and other protein molecules. Aptamers are practically non-immunogenic, chemically synthesized without the use of biological producers, and their antidotes can easily be created using complementary sequences. The review highlights reports devoted to the development of new anticoagulant aptamer-based medications. The most detailed studies, both preclinical and clinical (various phases of clinical trials), were performed in relation to the study of aptamers against vWF, factor IX and thrombin.

We used aptamers, which are functional equivalents of antibodies, in order to develop a nanosensor immunoassay system based on magnetic nanoparticles and a SQUID magnetometer. Selection was used to obtain DNA aptamers to interleukin-6; their affinity to the target protein was characterized by surface plasmon resonance. It was shown that the biotinylated aptamer binds to magnetic nanoparticles that were functionalized with streptavidin.

МЕТОД МАЛОУГЛОВОГО РЕНТГЕНОВСКОГО РАССЕЯНИЯ В ПРИМЕНЕНИИ К БИОЛОГИЧЕСКИМ МАКРОМОЛЕКУЛАМ

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.