Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors.

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

A responsive disulfide bond switch aptamer prodrug exhibiting enhanced stability and anticancer efficacy.

Aptamers have shown significant potential in treating diverse diseases. However, challenges such as stability and drug delivery limited their clinical application. In this paper, the development of AS1411 prodrug-type aptamers for tumor treatment was introduced. A Short oligonucleotide was introduced at the end of the AS1411 sequence with a disulfide bond as responsive switch. The results indicated that the aptamer prodrugs not only enhanced the stability of the aptamer against nuclease activity but also facilitated binding to serum albumin. Furthermore, in the reducing microenvironment of tumor cells, disulfide bonds triggered drug release, resulting in superior therapeutic effects in vitro and in vivo compared to original drugs. This paper proposes a novel approach for optimizing the structure of nucleic acid drugs, that promises to protect other oligonucleotides or secondary structures, thus opening up new possibilities for nucleic acid drug design.

A Poly Aptamer Encoded DNA Nanocatcher Informs Efficient Virus Trapping.

Broad-spectrum antiviral platforms are always desired but still lack the ability to cope with the threats to global public health. Herein, we develop a poly aptamer encoded DNA nanocatcher platform that can trap entire virus particles to inhibit infection with a broad antiviral spectrum. Ultralong single-stranded DNA (ssDNA) containing repeated aptamers was synthesized as the scaffold of a nanocatcher via a biocatalytic process, wherein mineralization of magnesium pyrophosphate on the ssDNA could occur and consequently lead to the formation of nanocatcher with interfacial nanocaves decorated with virus-binding aptamers. Once the viruses were recognized by the apatmers, they would be captured and trapped in the nanocaves via multisite synergistic interactions. Meanwhile, the size of nanocatchers was optimized to prevent their cellular uptake, which further guaranteed inhibition of virus infection. By taking SARS-CoV-2 variants as a model target, we demonstrated the broad virus-trapping capability of a DNA nanocatcher in engulfing the variants and blocking the infection to host cells.

The Evolution and Application of a Novel DNA Aptamer Targeting Bone Morphogenetic Protein 2 for Bone Regeneration.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

A new DNA aptamer which binds to SARS-CoV-2 spike protein and reduces pro-inflammatory response.

COVID-19 caused by SARS-CoV-2 spread rapidly around the world, endangering the health of people globally. The SARS-CoV-2 spike protein initiates entry into target cells by binding to human angiotensin-converting enzyme 2 (ACE2). In this study, we developed DNA aptamers that specifically bind to the SARS-CoV-2 spike protein, thereby inhibiting its binding to ACE2. DNA aptamers are small nucleic acid fragments with random structures that selectively bind to various target molecules. We identified nine aptamers targeting the SARS-CoV-2 spike protein using the systematic evolution of ligands by exponential enrichment (SELEX) method and selected three optimal aptamers by comparing their binding affinities. Additionally, we confirmed that the DNA aptamers suppressed pro-inflammatory cytokines induced by the SARS-CoV-2 spike protein in ACE2-overexpressing HEK293 cells. Overall, the DNA aptamer developed in this study has the potential to bind to the SARS-CoV-2 spike protein and inhibit or block its interaction with ACE2. Thus, our DNA aptamers can be used as new biological tools for the prevention and diagnosis of SARS-CoV-2 infection.

Innovative gelatin-based micelles with AS1411 aptamer targeting and reduction responsiveness for doxorubicin delivery in tumor therapy.

Herein, we constructed innovative reduction-sensitive and targeted gelatin-based micelles for doxorubicin (DOX) delivery in tumor therapy. AS1411 aptamer-modified gelatin-ss-tocopherol succinate (AGSST) and the control GSST without AS1411 modification were synthesized and characterized. Antitumor drug DOX-containing AGSST (AGSST-D) and GSST-D nanoparticles were prepared, and their shapes were almost spherical. Reduction-responsive characteristics of DOX release in vitro were revealed in AGSST-D and GSST-D. Compared with non-targeted GSST-D, AGSST-D demonstrated better intracellular uptake and stronger cytotoxicity against nucleolin-overexpressed A549 cells. Importantly, AGSST-D micelles showed more effective killing activity in A549-bearing mice than GSST-D and DOX⋅HCl. It was revealed that AGSST-D micelles had no obvious systemic toxicity. Overall, AGSST micelles would have the potential to be an effective drug carrier for targeted tumor therapy.

An anti-GD2 aptamer-based bifunctional spherical nucleic acid nanoplatform for synergistic therapy targeting MDM2 for retinoblastoma.

Retinoblastoma (RB) is a type of pediatric solid tumor in the fundus. The lack of precision therapies combined with the difficulty of delivering small interfering RNA (siRNA) into the eyes means that there is currently no nucleic acid-based therapy for RB in clinical practice. Here, we reported on anti-GD2 and glutathione-responsive spherical nucleic acids (SNAs), loaded with siRNA and the inhibitor NVP-CGM097, which jointly blocked the oncogenic factor n in RB cells (Y79 and WERI-RB-1). The SNAs were formed through the self-assembly of bifunctional cholesterol amphiphiles containing aptamers that specifically targeted GD2-positive RB cells, allowing for the formation of an SNA with a dense DNA shell. The aptamer/siRNA component functioned both as a carrier and a payload, enhancing the specific recognition and delivery of both components and constituting an active agent for MDM2 regulation. Following SNA endocytosis by RB cells, siRNA and NVP-CGM097 were released from the SNA particles by glutathione, which synergistically blocked the MDM2-p53 pathway, increasing p53 protein content and inducing cell apoptosis. This study showed a potent antitumor effect following intravitreal injection of SNAs in Y79 tumor-bearing mice through clinical manifestation and tumor pathological analysis. In hematological analysis and hepatotoxicity assays, SNAs were safer for mice than melphalan, the favored drug for treating RB in clinical practice. Our results illustrated the potential of intravitreally injected SNAs as a precision medicine for treating RB.

Construction of aptamer-siRNA chimera and glutamine modified carboxymethyl-ß-cyclodextrin nanoparticles for the combination therapy against lung squamous cell carcinoma.

Combination therapy has become the most important treatment for advanced non-small cell lung cancer (NSCLC), which can significantly improve the prognosis of patients. However, poor targeting and adverse reactions limited its clinical application. Here, we constructed an AS1411 aptamer-programmed cell death ligand-1 (PD-L1) siRNA chimera/polyethylenimine/glutamine/ß-cyclodextrin/doxorubicin (Chimera/ PEI/Gln/ß-CD/DOX) nanoparticle for the combination therapy (chemotherapy combined with immunotherapy). Scanning electron microscopy showed that PEI/Gln/ß-CD/DOX nanoparticle was conical, with a diameter of about 250-500 nm. AS1411 aptamer-PD-L1 siRNA chimera can effectively bind NSCLC cells and inhibit PD-L1 expression, further activating T cells and CD8+T cells. Glutamine modification effectively promoted the doxorubicin uptake by cancer cells and induced their apoptosis. Animal experiments showed that our nanoparticles effectively treated the transplanted tumor, and the adverse reactions were reduced. Compared with the Aptamer/ß-CD/DOX group, the volume and ki-67 index of transplanted tumors in the Chimera/ß-CD/DOX group were significantly decreased, while the apoptosis ratio was increased. Immunohistochemical results showed that Compared with the Aptamer/ß-CD/DOX group, the number of T cells and CD8+T cells in the Chimera/ß-CD/DOX group was increased by 1.34 and 1.41 times. Glutamine modification enhanced the chemotherapeutic efficacy and anti-tumor immune response in vivo. Our study provided a new method for the combination therapy of lung squamous cell carcinoma.

[Covalently conjugated DNA aptamer with doxorubicin as in vitro model for effective targeted drug delivery to human glioblastoma tumor cells]. / Kovalentno kon"yugirovannyi DNK-aptamer s doksorubitsinom kak in vitro model' effektivnogo adresnogo vozdeistviya na opukholevye kletki glioblastomy cheloveka.

Targeted delivery of chemotherapeutic agents with aptamers is a very effective method increasing therapeutic index compared to non-targeted drugs. OBJECTIVE: To study the effectiveness of in vitro therapeutic effect of covalently conjugated GR20 DNA aptamer with doxorubicin on glioblastoma cells compared to reference culture of human fibroblasts. MATERIAL AND METHODS: A Sus/fP2 cell culture was obtained from glioblastoma tissue sample to analyze the effectiveness of conjugate. A linear culture of human dermal fibroblasts (mesenchymal stem cells) DF1 was used as a control. To assess antiproliferative activity of covalently conjugated GR20 aptamer with doxorubicin, we used the MTS test. The Cell Index was measured using the xCelligence S16 cell analyzer assessing viability of cell cultures by recording changes in real time. RESULTS: Human glioblastoma Sus/fP2 cells reduce own proliferative potential by 80% when exposed to doxorubicin (0.5 µM, 72 hours, MTS test), by 9% when exposed to GR20 aptamer (10 µM, 72 hours, MTS test) and by 26% when exposed to covalently conjugated DOX-GR20 (0.5 µM, 72 hours, MTS test). A long-term study of proliferative potential of Sus/fP2 cells on the xCelligence S16 analyzer revealed a significant decrease in the number of cells under the effect of doxorubicin and covalently conjugated DOX-GR20. Effectiveness of covalently conjugated DOX-GR20 is halved. GR20 aptamer at a concentration of 10 µM and its conjugate with doxorubicin DOX-GR20 at a concentration of 1 µM have no negative effect on cells of the control culture of DF1 fibroblasts, while doxorubicin is toxic for these cells. MTS test and xCelligence S16 cell analyzer found no decrease in metabolic activity of DF1 cells and their ability to proliferate. CONCLUSION: We established obvious antiproliferative effect of covalent conjugate DOX-GR20 on continuous human glioblastoma cell culture Sus/fP2 without toxic effect on the reference culture (dermal fibroblasts DF1).

Directing in Vitro Selection towards G-quadruplex-forming Aptamers to Inhibit HMGB1 Pathological Activity.

In the search for novel, effective inhibitors of High-Mobility Group Box1 (HMGB1)-a protein involved in various inflammatory and autoimmune diseases as well as in cancer-we herein discovered a set of anti-HMGB1 G-quadruplex(G4)-forming aptamers by using an in vitro selection procedure applied to a doped library of guanine-rich oligonucleotides. The selected DNA sequences were then studied in a pseudo-physiological buffer mimicking the extracellular medium, where HMGB1 exerts its pathological activity, using spectroscopic, electrophoretic, and chromatographic techniques. All the oligonucleotides proved to fold into monomeric G4s and in some cases also dimeric species, stable at physiological temperature. Remarkably, the protein preferentially recognized the sequences forming dimeric parallel G4 structures, as evidenced by a properly designed chemiluminescent binding assay which also highlighted a good selectivity of these aptamers for HMGB1. Moreover, all aptamers showed anti-HMGB1 activity, inhibiting protein-induced cell migration. The acquired data allowed identifying L12 as the best anti-HMGB1 aptamer, featured by high thermal and enzymatic stability, no toxicity at least up to 5 µM concentration on healthy cells, along with potent anti-HMGB1 activity (IC50 ca. 28 nM) and good binding affinity for the protein, thus indicating it as a very promising lead candidate for in vivo studies.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Blocking pathogenic Leptospira invasion with aptamer molecules targeting outer membrane LipL32 protein.

This study aimed to develop aptamers targeting LipL32, a most abundant lipoprotein in pathogenic Leptospira, to hinder bacterial invasion. The objectives were to identify high-affinity aptamers through SELEX and evaluate their specificity and inhibitory effects. SELEX was employed to generate LipL32 aptamers (L32APs) over 15 rounds of selection. L32APs' binding affinity and specificity for pathogenic Leptospira were assessed. Their ability to inhibit LipL32-ECM interaction and Leptospira invasion was investigated. Animal studies were conducted to evaluate the impact of L32AP treatment on survival rates, Leptospira colonization, and kidney damage. Three L32APs with strong binding affinity were identified. They selectively detected pathogenic Leptospira, sparing non-pathogenic strains. L32APs inhibited LipL32-ECM interaction and Leptospira invasion. In animal studies, L32AP administration significantly improved survival rates, reduced Leptospira colonies, and mitigated kidney damage compared to infection alone. This pioneering research developed functional aptamers targeting pathogenic Leptospira. The identified L32APs exhibited high affinity, pathogen selectivity, and inhibition of invasion and ECM interaction. L32AP treatment showed promising results, enhancing survival rates and reducing Leptospira colonization and kidney damage. These findings demonstrate the potential of aptamers to impede pathogenic Leptospira invasion and aid in recovery from Leptospira-induced kidney injury (190 words).

Reversible Aptamer Staining, Sorting, and Cleaning of Cells (Clean FACS) with Antidote Oligonucleotide or Nuclease Yields Fully Responsive Cells.

The ability to reverse the binding of aptamers to their target proteins has received considerable attention for developing controllable therapeutic agents. Recently, use of aptamers as reversible cell-sorting ligands has also sparked interest. Antibodies are currently utilized for isolating cells expressing a particular cell surface receptor. The inability to remove antibodies from isolated cells following sorting greatly limits their utility for many applications. Previously, we described how a particular aptamer-antidote oligonucleotide pair can isolate cells and clean them. Here, we demonstrate that this approach is generalizable; aptamers can simultaneously recognize more than one cell type during fluorescent activated cell sorting (FACS). Moreover, we describe a novel approach to reverse aptamer binding following cell sorting using a nuclease. This alternative strategy represents a cleaning approach that does not require the generation of antidote oligonucleotides for each aptamer and will greatly reduce the cost and expand the utility of Clean FACS.

Targeting vimentin: a multifaceted approach to combatting cancer metastasis and drug resistance.

This comprehensive review explores vimentin as a pivotal therapeutic target in cancer treatment, with a primary focus on mitigating metastasis and overcoming drug resistance. Vimentin, a key player in cancer progression, is intricately involved in processes such as epithelial-to-mesenchymal transition (EMT) and resistance mechanisms to standard cancer therapies. The review delves into diverse vimentin inhibition strategies. Precision tools, including antibodies and nanobodies, selectively neutralize vimentin's pro-tumorigenic effects. DNA and RNA aptamers disrupt vimentin-associated signaling pathways through their adaptable binding properties. Innovative approaches, such as vimentin-targeted vaccines and microRNAs (miRNAs), harness the immune system and post-transcriptional regulation to combat vimentin-expressing cancer cells. By dissecting vimentin inhibition strategies across these categories, this review provides a comprehensive overview of anti-vimentin therapeutics in cancer treatment. It underscores the growing recognition of vimentin as a pivotal therapeutic target in cancer and presents a diverse array of inhibitors, including antibodies, nanobodies, DNA and RNA aptamers, vaccines, and miRNAs. These multifaceted approaches hold substantial promise for tackling metastasis and overcoming drug resistance, collectively presenting new avenues for enhanced cancer therapy.

Aptamer-Modified Tetrahedral DNA Nanostructures as Drug Delivery System for Cancer Targeted Therapy.

Improving the selective delivery and uptake efficiency of chemotherapeutic drugs remains a challenge for cancer-targeted therapy. In this work, a DNA tetrahedron is constructed as a targeted drug delivery system for efficient delivery of doxorubicin (Dox) into cancer cells. The DNA tetrahedron is composed of a tetrahedral DNA nanostructure (TDN) with two strands of AS1411 aptamer as recognition elements which can target the nucleolin protein on the cell membrane of cancer cells. The prepared DNA tetrahedron has a high drug-loading capacity and demonstrates pH-responsive Dox release properties. This enables efficient delivery of Dox into targeted cancer cells while reducing side effects on nontarget cells. The proposed drug delivery system exhibits significant therapeutic efficacy in vitro compared to free Dox. Accordingly, this work provides a good paradigm for developing a targeted drug delivery system for cancer therapy based on DNA tetrahedrons.

Aptamer-based diagnostic and therapeutic approaches for animal viruses: A review.

Animal diseases often have significant consequences due to the unclear and time-consuming diagnosis process. Furthermore, the emergence of new viral infections and drug-resistant pathogens has further complicated the diagnosis and treatment of viral diseases. Aptamers, which are obtained through systematic evolution of ligands by exponential enrichment (SELEX) technology, provide a promising solution as they enable specific identification and binding to targets, facilitating pathogen detection and the development of novel therapeutics. This review presented an overview of aptasensors for animal virus detection, discussed the antiviral activity and mechanisms of aptamers, and highlighted advancements in aptamer-based antiviral research following the COVID-19 pandemic. Additionally, the challenges and prospects of aptamer-based virus diagnosis and treatment research were explored. Although this review was not exhaustive, it offered valuable insights into the progress of aptamer-based antiviral drug research, target mechanisms, as well as the development of novel antiviral drugs and biosensors.

Isolation and Identification of the High-Affinity DNA Aptamer Target to the Brain-Derived Neurotrophic Factor (BDNF).

Aptamers are functional oligonucleotide ligands used for the molecular recognition of various targets. The natural characteristics of aptamers make them an excellent alternative to antibodies in diagnostics, therapeutics, and biosensing. DNA aptamers are mainly single-stranded oligonucleotides (ssDNA) that possess a definite binding to targets. However, the application of aptamers to the fields of brain health and neurodegenerative diseases has been limited to date. Herein, a DNA aptamer against the brain-derived neurotrophic factor (BDNF) protein was obtained by in vitro selection. BDNF is a potential biomarker of brain health and neurodegenerative diseases and has functions in the synaptic plasticity and survival of neurons. We identified eight aptamers that have binding affinity for BDNF from a 50-nucleotide library. Among these aptamers, NV_B12 showed the highest sensitivity and selectivity for detecting BDNF. In an aptamer-linked immobilized sorbent assay (ALISA), the NV_B12 aptamer strongly bound to BDNF protein, in a dose-dependent manner. The dissociation constant (Kd) for NV_B12 was 0.5 nM (95% CI: 0.4-0.6 nM). These findings suggest that BDNF-specific aptamers could be used as an alternative to antibodies in diagnostic and detection assays for BDNF.

Using an RNA Aptamer to Inhibit the Action of Effector Proteins of Plant Pathogens.

In previous work, we experimentally demonstrated the possibility of using RNA aptamers to inhibit endogenous protein expression and their function within plant cells In the current work, we show that our proposed method is suitable for inhibiting the functions of exogenous, foreign proteins delivered into the plant via various mechanisms, including pathogen proteins. Stringent experimentation produced robust RNA aptamers that are able to bind to the recombinant HopU1 effector protein of P. syringae bacteria. This research uses genetic engineering methods to constitutively express/transcribe HopU1 RNA aptamers in transgenic A. thaliana. Our findings support the hypothesis that HopU1 aptamers can actively interfere with the function of the HopU1 protein and thereby increase resistance to phytopathogens of the genus P. syringae pv. tomato DC 3000.

Tools and techniques for the discovery of therapeutic aptamers: recent advances.

INTRODUCTION: The pursuit of novel therapeutic agents for serious diseases such as cancer has been a global endeavor. Aptamers characteristic of high affinity, programmability, low immunogenicity, and rapid permeability hold great promise for the treatment of diseases. Yet obtaining the approval for therapeutic aptamers remains challenging. Consequently, researchers are increasingly devoted to exploring innovative strategies and technologies to advance the development of these therapeutic aptamers. AREAS COVERED: The authors provide a comprehensive summary of the recent progress of the SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique, and how the integration of modern tools has facilitated the identification of therapeutic aptamers. Additionally, the engineering of aptamers to enhance their functional attributes, such as inhibiting and targeting, is discussed, demonstrating the potential to broaden their scope of utility. EXPERT OPINION: The grand potential of aptamers and the insufficient development of relevant drugs have spurred countless efforts for stimulating their discovery and application in the therapeutic field. While SELEX techniques have undergone significant developments with the aid of advanced analysis instruments and ingeniously updated aptameric engineering strategies, several challenges still impede their clinical translation. A key challenge lies in the insufficient understanding of binding conformation and susceptibility to degradation under physiological conditions. Despite the hurdles, our opinion is optimistic. With continued progress in overcoming these obstacles, the widespread utilization of aptamers for clinical therapy is envisioned to become a reality soon.

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote.

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.