The affinity of nucleic acid aptamers isolated in vitro via Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is often limited because the entire potential sequence space cannot be screened. In this study, we introduce Motif-SELEX, a novel method that enables the optimization of existing underperforming aptamers by generating libraries that broadly represent both the sequence and length variations of the parent sequence. This approach enables the isolation of sequences with improved affinity without the biases and limitations of traditional mutagenesis methods like doped SELEX and error-prone PCR. As a demonstration, we applied Motif-SELEX to a DNA-based morphine aptamer and a 2' fluoro- and methoxy-RNA-based apixaban aptamer, discovering new, better-performing sequences with differing random domain lengths from their parents and up to 10-fold improvements in affinity. These new sequences would be inaccessible to traditional post-SELEX methods. Critically, our analysis of Motif-SELEX pools also enabled us to identify sequence and structural elements crucial for target binding and to predict secondary and tertiary structures for a given aptamer family─even when those structures involve noncanonical nucleotide interactions. We believe that Motif-SELEX offers an effective and generalizable solution for optimizing the structure and binding properties of functional nucleic acid molecules for diverse applications.

Unfractionated heparin (UFH), designated as an essential medicine by the World Health Organization (WHO), is indispensable in cardiac surgery and various clinical applications. However, its production depends on the farming of over a billion large animals annually, posing sustainability challenges, especially amidst increasing efforts to mitigate greenhouse gas (GHG) emissions by reducing animal farming. The rising demand for UFH has outpaced its supply, and previous attempts to develop synthetic alternatives have fallen short because of their limited potency and reversibility. Here, HD1-12dmA-DAB is presented, a novel synthetic anticoagulant developed through chemical conjugation of a thrombin exosite-binding aptamer (HD1) with a thrombin active-site inhibitor (dabigatran). By optimizing dual-site synergistic binding, HD1-12dmA-DAB achieves an enhancement of over three orders of magnitude in thrombin-binding affinity and specificity compared to HD1 alone. It also demonstrates superior plasma stability and anticoagulant efficacy comparable to UFH, as validated in in vitro, in vivo, and ex vivo clotting models. This breakthrough highlights a promising step toward a sustainable, animal-free alternative to UFH, addressing the growing clinical demand and advancing environmental sustainability objectives.

Background and purpose:Unfractionated heparin (UFH) is the most commonly utilized rapid-onset anticoagulant, valued for its potency and reversibility with protamine. However, UFH and protamine are associated with significant side effects, including increased morbidity and mortality, and concerns about sustainability due to the environmental impact of large-scale pig farming for heparin production. This study evaluates an alternative anticoagulant strategy using a factor IXa (FIXa) aptamer paired with a matched oligonucleotide antidote, comparing its efficacy and safety to heparin-protamine in a rat extracorporeal membrane oxygenation (ECMO) model. Methods:Twenty-four Sprague-Dawley rats were randomized into two groups: one receiving heparin (600 IU/kg) and protamine (1 mg/100 IU heparin), and the other receiving a cholesterol-modified FIXa aptamer (10 mg/kg) and its antidote (50 mg/kg). Coagulation parameters, platelet counts, inflammatory markers, cardiac function, and histopathology were assessed during and after 60 minutes of ECMO. Results:The FIXa aptamer effectively maintained circuit patency without clot formation, comparable to heparin. The antidote rapidly reversed the aptamer's anticoagulant activity, similar to protamine's reversal of heparin. Notably, the aptamer-antidote group demonstrated superior outcomes, including improved mean arterial pressure (58 ± 6 mmHg vs. 54 ± 3 mmHg at 30 minutes; 59 ± 8 mmHg vs. 51 ± 5 mmHg at 3 hours post-ECMO) and cardiac function (shortening fraction: 60 ± 16% vs. 42 ± 8%; P = 0.01). Additionally, the aptamer group exhibited better platelet preservation (platelet count decrease: -288,000 ± 121,000/μL vs. -404,000 ± 89,000/μL; P = 0.03). Inflammatory profiles were similar between groups, except for a transient increase in interleukins 10 (IL-10) in the aptamer group. Histopathological analysis revealed no significant differences in myocardial lesions. Conclusions:The antidote-controlled anti-FIXa aptamer represents an alternative anticoagulant strategy that may prove useful for managing patients with a history of heparin-induced thrombocytopenia (HIT) and myocardial dysfunction associated with protamine administration.

Clustered regularly interspaced short palindromic repeats-based editing is inefficient at over two-thirds of genetic targets. A primary cause is ribonucleic acid (RNA) misfolding that can occur between the spacer and scaffold regions of the gRNA, which hinders the formation of functional Cas9 ribonucleoprotein (RNP) complexes. Here, we uncover hundreds of highly efficient gRNA variant scaffolds for Staphylococcus aureus (Sa)Cas9 utilizing an innovative binding and ligand activation driven enrichment (BLADE) methodology, which leverages asymmetrical product dissociation over rounds of evolution. SaBLADE-derived gRNA scaffolds contain 7%-42% of nucleotide variation relative to wild type. gRNA variants are able to improve gene editing efficiency at all targets tested, and they achieve their highest levels of editing improvement (>400%) at the most challenging DNA target sites for the wild-type SaCas9 gRNA. This arsenal of SaBLADE-derived gRNA variants showcases the power and flexibility of combinatorial chemistry and directed evolution to enable efficient gene editing at challenging, or previously intractable, genomic sites.

Abstract: In May 2024, the Division of Blood Diseases and Resources of the National Heart, Lung, and Blood Institute (NHLBI) hosted a hybrid workshop on “Extracorporeal membrane oxygenation (ECMO)-induced coagulopathy: strategic initiatives for research and clinical practice.” The event brought together clinicians, scientists, bioengineers, and policymakers to address the challenges of ECMO-associated coagulopathy and explore novel therapeutic approaches. Through expert presentations and collaborative discussions, the workshop focused on innovative anticoagulation strategies, precision medicine, and advanced diagnostics to enhance patient care. The discussions also identified critical research gaps and opportunities for future interdisciplinary collaboration. This summary reviews the current state of knowledge and outlines future research directions for improving ECMO-induced coagulopathy management.

Background: Unfractionated heparin (UFH), the main anticoagulant used in heart surgeries, is associated with adverse effects such as bleeding and heparin-induced thrombocytopenia. Anticoagulant aptamers targeting coagulation proteases have advantages such as low immunogenicity, low bleeding risk, and reversibility by complementary oligonucleotides. However, the potency of aptamers is often limited as they do not directly inhibit the catalytic sites on their targeted proteases. The natural anticoagulant hirudin achieves potent activity by inhibiting both thrombin’s exosite and active site. We hypothesize that hirudin-like protease inhibitors can be generated de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors to create EXACT inhibitors that are much more potent than the small molecule inhibitors or aptamers alone. Methods: Protease inhibition by EXACT inhibitors were characterized by fluorogenic substrate cleavage assays. The anticoagulation activity of EXACT inhibitors was assessed in normal pooled plasma or whole blood using the standard clinical clotting assays: prothrombin time (PT), activated partial thromboplastin time (aPTT), and active clotting time (ACT). The clinical potential of the EXACT inhibitor was evaluated in an ex vivo Extracorporeal Membrane Oxygenation (ECMO) circuit that mimics the procoagulant environment in many cardiovascular surgical settings. Results: An EXACT inhibitor termed HD1-12dmA-DAB was synthesized by attaching the DNA aptamer HD1, that binds to thrombin exosite I, through a poly-adenosine linker to the thrombin active site inhibitor dabigatran (DAB). The EXACT inhibitor showed stronger thrombin inhibition (IC 50 = 0.2 nM) than DAB (IC 50 = 80 nM) or HD1 (IC 50 > 1 µM) alone. HD1-12dmA-DAB (2 µM) also achieved equal or higher anticoagulant activity than UFH (5U/mL) in all clotting assays and showed anticoagulation efficacy rivaling UFH in the ex vivo ECMO circuit with minimal clot observed over 120 min. Notably, the EXACT inhibitor’s effect can be rapidly reversed by an antidote oligo. We also used the same strategy to develop a potent EXACT inhibitor against factor Xa. Conclusions: EXACT inhibitors demonstrate extraordinary anticoagulation activity as potential rapid onset anticoagulants to support cardiovascular procedures. Using this generalizable strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes for numerous therapeutic applications.

During extracorporeal membrane oxygenation (ECMO) support, the high shear stress in the ECMO circuit results in increased proteolysis of von Willebrand factor (VWF), loss of VWF high-molecular-weight multimers, and impaired ability to bind to platelets and collagen. These structural changes in VWF are consistent with acquired von Willebrand syndrome (AVWS) type 2A and may contribute to the bleeding diathesis frequently observed in ECMO patients. We performed a systematic review of all clinical studies evaluating the prevalence and associated outcomes of AVWS in ECMO patients. Our findings suggest that almost all ECMO patients develop partial or complete loss of VWF high-molecular-weight multimers within a few hours of device implantation. The AVWS persists as long as the patient is supported by ECMO. Weaning from ECMO rapidly and completely resolves the AVWS. Nevertheless, few studies have reported bleeding outcomes in ECMO patients with AVWS, and the extent to which AVWS contributes to the bleeding diathesis during ECMO support cannot be determined by current evidence. Data supporting the use of VWF concentrates to prevent bleeding complications in ECMO patients remain limited.

Background: Serine proteases of blood coagulation are specifically modulated by cofactors to enable timely hemostatic response without inducing thrombosis. Coagulation factor IXa, a protease component of the enzymic complex called 'intrinsic Xase', is an enigmatic enzyme due to extremely low catalytic activity in the free state. Although its protease domain has the same architecture of an activated trypsin-like protease as homologous coagulation enzymes thrombin and factor Xa, the mechanism of factor IXa function remains elusive. Objective: to study the conformational ensemble of factor IXa and reveal structural transitions enabling the gain and loss of the catalytic function. Methods: X-ray crystallography was used to resolve the structures of recombinant human factor IXa (S195A) with truncated N-terminal domains that was purified from stable AV12 cell clones. Binding affinity to biotinylated anti-IXa aptamer was measured by bio-layer interferometry in conditions with negligible mass-transport effects. Affinity of the IXa-aptamer interaction in solution was measured by fluorescence anisotropy. Active site function was assessed by kinetic measurements of fluorogenic substrate cleavage and by formation of covalent complex with antithrombin. Results: Structure of the apo-enzyme is resolved at 1.9 Å. Co-crystal structure with anti-IXa RNA aptamer having anticoagulant effect is resolved at 2.6 Å. In the apo-state, the active site of factor IXa is found in the latent conformation, with W215 side chain interfering with the entrance into S1 substrate-binding pocket. In comparison, structures of apo- thrombin and factor Xa both exhibit the open conformation of the S1 pocket, which explains a uniquely low catalytic turnover by IXa in comparison to homologous proteases. The co-crystal structure of IXa-aptamer complex shows that the aptamer does not occlude the active site and binds the protease domain in the remote exosite region, which was previously implicated in binding cofactor VIIIa and anticoagulant drug heparin. The structure shows that residues K132, L162, R165, R233 of factor IXa are important for the interaction with aptamer. Together with previous mutagenesis data, this indicates the determinants of sub-nanomolar affinity and exclusive specificity of the aptamer to IXa. Comparison of the active site conformations in apo- vs. aptamer-bound vs. substrate-bound states of factor IXa demonstrates the structural mechanism explaining the inhibitory action of the aptamer. The RNA aptamer acts allosterically by causing a 7-Å displacement of W215 that leads to the closure of S1 pocket. These structural rearrangements correlate with the aptamer-mediated inhibition of both substrate cleavage and active site-mediated interaction with antithrombin. Conclusions: This work provides the first structural evidence of allosteric control of enzymatic activity in factor IXa. The molecular surface occupied by RNA aptamer reveals an important exosite for the specific recognition and regulation of the enzymic complex assembly and catalysis. Supported by the known exosite mutations causing either bleeding or thrombotic phenotype, these findings suggest the importance of the exosite to allosterically modulate active site function of factor IXa and control the hemostatic balance. The work was supported by grant P01HL139420 from the NHLBI to S.K. and B.A.S. X-ray diffraction data were collected at the 17-ID-1 beamline at the NSLS II, a U.S. DOE Office of Science User Facility and the NE-CAT 24-ID-C beamline funded by NIH grant GM103403 with a Pilatus 6M detector funded by a NIH-ORIP HEI grant (RR029205).

Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp 215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

Secreted microRNAs (miRNAs) have been detected in various body fluids including the cerebrospinal fluid, yet their direct role in regulating synaptic transmission remains uncertain. We found that intrathecal injection of low dose of let-7b (1 mu g) induced short-term (<24 hours) mechanical allodynia and heat hyperalgesia, a response that is compromised in Tlr7-/- or Trpa1-/- mice. Ex vivo and in vivo calcium imaging in GCaMP6-report mice revealed increased calcium signal in spinal cord afferent terminals and doral root ganglion/dorsal root ganglia neurons following spinal perfusion and intraplantar injection of let-7b. Patch-clamp recordings also demonstrated enhanced excitatory synaptic transmission (miniature excitatory postsynaptic currents [EPSCs]) in spinal nociceptive neurons following let-7b perfusion or optogenetic activation of axonal terminals. The elevation in spinal calcium signaling and EPSCs was dependent on the presence of toll-like receptor-7 (TLR7) and transient receptor potential ion channel subtype A1 (TRPA1). In addition, endogenous let-7b is enriched in spinal cord synaptosome, and peripheral inflammation increased let-7b in doral root ganglion/dorsal root ganglia neurons, spinal cord tissue, and the cerebrospinal fluid. Notably, let-7b antagomir inhibited inflammatory pain and inflammation-induced synaptic plasticity (EPSC increase), suggesting an endogenous role of let-7b in regulating pain and synaptic transmission. Furthermore, intrathecal injection of let-7b, at a higher dose (10 mu g), induced persistent mechanical allodynia for >2 weeks, which was abolished in Tlr7(-/- )mice. The high dose of let-7b also induced microgliosis in the spinal cord. Of interest, intrathecal minocycline only inhibited let-7b-induced mechanical allodynia in male but not female mice. Our findings indicate that the secreted microRNA let-7b has the capacity to provoke pain through both neuronal and glial signaling, thereby establishing miRNA as an emerging neuromodulator.

Lymphocyte telomere length (TL) is highly variable and shortens with age. Short telomeres may impede TL-dependent T-cell clonal expansion with viral infection. As SARS-CoV-2 infection can induce prolonged and severe T-cell lymphopenia, infected adults, and particularly older adults with short telomeres, may display severe T-cell lymphopenia. To examine the relationship between T-cell TL parameters and T-cell counts, we studied 40 patients hospitalized with severe COVID-19. T-cells were isolated from lymphocytes, counted using flow cytometry, and their TL parameters were measured using the Telomere Shortest Length Assay. The cohort (median age = 62 years, 27% female) was racially and ethnically diverse (33% White, 35% Black, and 33% Other). On intensive care unit study day 1, T-cell count (mean=1.03 x109/L) was inversely related to age (p=0.007) and higher in females than males (p=0.025). Mean TL was 3.88 kilobases (kb), and 45.3% of telomeres were shorter than 3 kb. Using multiple regression analysis and adjusting for age and sex, T-cell count decreased with increased proportion of T-cell telomeres shorter than 3 kb (p=0.033) and increased with mean TL (p=0.052). Our findings suggest an association between the buildup of short telomeres within T-cells and explain in part reduced peripheral blood T-cell counts in patients with severe COVID-19. Shortened T-cell telomeres may be a risk factor for COVID-19-associated T-cell lymphopenia.

Background and purposePosterior circulation strokes, accounting for 20% of acute ischemic strokes, significantly contribute to morbidity and mortality. Fibrinolysis by rtPA improves outcomes in stroke but the risk of intracranial hemorrhage limits benefit. Arterial recanalization of basilar artery occlusion by thrombolysis or endovascular thrombectomy improves outcomes in posterior circulation strokes. This study investigates a VWF-targeting RNA aptamer as a safer and more effective alternative to rtPA in a canine model.Materials and methodsAutologous clots were placed into the basilar artery to induce stroke in 24 beagles. To compare reperfusion, 0.9 mg/kg rtPA, 0.5 mg/kg BB-031, or vehicle were administered 60 min after the initiation of occlusion. Digital subtraction angiography, laser speckle imaging and magnetic resonance imaging were used to assess recanalization, reperfusion and infarct volume, respectively.ResultsTreatment with BB-031 resulted in recanalization of the posterior circulation on digital subtraction angiography with no evidence of microembolism assessed at sacrifice. 66.5% of animals treated with BB-031 resulted in reperfusion with a TICI score of ≥1 whereas vehicle remained at TICI score 0 as did all but one rtPA animal at sacrifice. Improved perfusion was seen in the basilar artery and surrounding blood vessels visualized through the cranial window with laser speckle imaging to ~47% of its original baseline in BB-031 group compared to rtPA at 37% and vehicle at 22%. Finally, BB-031-treatment resulted in an approximate 32% mean infarct volume, significantly smaller on magnetic resonance imaging compared to 56% in vehicle treated and 48% with rtPA treatment.ConclusionTargeted inhibition of VWF by BB-031 increased recanalization and reperfusion, and reduced infarct volume in a canine model of BAO stroke. It represents a promising target based on preliminary results for treating acute ischemic stroke.

BACKGROUND:Andexanet alfa (andexanet) is the only Food and Drug Administration-approved antidote for direct FXa (factor Xa) inhibitors but has been reported to cause resistance to unfractionated heparin (UFH). This has delayed anticoagulation for procedures requiring cardiopulmonary bypass. The mechanism, andexanet and UFH dose dependence, and thrombotic risk of andexanet-associated heparin resistance are unknown.METHODS:The effect of andexanet in vitro was determined using activated clotting times and thromboelastography. Ex vivo cardiopulmonary bypass circuits were used to determine whether andexanet impaired anticoagulation for extracorporeal circulation. Kinetics of AT (antithrombin) inhibition of FXa and thrombin were measured in the presence of andexanet. Equilibrium modeling and thrombin generation assay validation were used to predict the role of andexanet, AT, and UFH concentrations in andexanet-associated heparin resistance.RESULTS:Andexanet prevented UFH-mediated prolongation of activated clotting times and thromboelastography times. At lower concentrations of andexanet, heparin resistance could be overcome with suprapharmacologic doses of UFH, but not at higher andexanet concentrations. Andexanet rendered standard doses of UFH inadequate to prevent circuit thrombosis, and suprapharmacologic UFH doses were only partially able to overcome this. Scanning electron microscopy demonstrated coagulation activation in circuits. Andexanet prevented UFH enhancement of AT-mediated inhibition of FXa and thrombin. Equilibrium modeling and thrombin generation assay validation demonstrated that andexanet creates a triphasic equilibrium with UFH and AT: initial UFH unresponsiveness, normal UFH responsiveness when andexanet is depleted, and finally AT depletion. Sufficient cardiopulmonary bypass heparinization can only occur at low therapeutic andexanet doses and normal AT levels. Higher andexanet doses or AT deficiency may require high UFH doses and potentially AT supplementation.CONCLUSIONS:Andexanet causes heparin resistance due to redistribution of UFH-bound AT. If andexanet cannot be avoided before heparinization and direct thrombin inhibitors are undesirable, our in vitro study suggests excess UFH should be considered as a potential strategy before AT supplementation.

Severe COVID-19 carries a high morbidity and mortality. Previous studies have shown an association between COVID-19 severity and SARS-CoV-2 viral load (VL). We sought to measure VL in multiple compartments (urine, plasma, lower respiratory tract) in patients admitted to the intensive care unit (ICU) with severe COVID-19 pneumonia and correlate with clinical outcomes. Plasma, urine, and endotracheal aspirate (ETA) samples were obtained on days 1, 3, 7, 14, and 21 from subjects admitted to the ICU with severe COVID-19. VL was measured via reverse transcriptase polymerase chain reaction. Clinical data was collected from the electronic health record. Grouped comparisons were performed using Student’s t-test or 1-way ANOVA. Linear regression was used to correlate VL from different compartments collected at the same time. Logistic regression was performed to model ventilator-freedom at 28 days as a function of peak plasma VL. We enrolled 57 subjects with severe COVID-19 and measured VL in plasma (n = 57), urine (n = 25), and ETA (n = 34). Ventilator-associated pneumonia developed in 63

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. Inspired by the biologics of hematophagous organisms such as leeches, the authors in this work design and create inhibtors of thrombin and factor Xa by linking exosite-binding aptamers with small molecule active site inhibtors. They coin these inhibitors EXACT inhibitors.

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.

Cell isolation from complex mixtures is a key step in many clinical and research applications, but standard isolation methods may affect the cell’s biology and are difficult to reverse. Here, we present a method to isolate and restore cells to their native state using an aptamer that binds epidermal growth factor receptor (EGFR+)cells and a complementary antisense oligonucleotide to reverse binding.

Background : Coagulation cascade cofactors are essential proteins that control homeostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging due to their lack of an active site. Aims : Development of FV/FVa and FVIII-inhibiting aptamers as potent and reversible anticoagulants that can be used to aid (or replace) heparin for therapeutic anticoagulation. Reveal new mechanisms of cofactor inhibition. Methods : 2 ' F modified RNA Aptamers were identified via in vitro systematic evolution of ligand by exponential enrichment (SELEX) and post-SELEX truncation. Aptamer affinity was characterized by nitrocellulose filter binding assays and surface plasmon resonance. Aptamer bioactivity was analyzed using clotting assays and biochemical assays. Aptamers ' binding mechanism was determined using fluorescent anisotropy, fluorescence resonance energy transfer, dynamic light scattering, and molecular modeling. Results : An aptamer termed T18.3 binds to both human FV/FVa with K D ∼10 nM and prolongs aPTT in normal human plasma by 3.5-fold at a concentration of 500 nM. The aptamer inhibits membrane docking of FVa by binding to FV/FVa light chain, thereby reduces prothrombinase assembly and thrombin generation. Notably, the aptamer showed similar anticoagulant potency in normal, FV Leiden, and COVID-19 patient plasma. The aptamer also synergistically inhibits clotting with enoxaparin and can be rapidly reversed by protamine in in vitro assays. Another aptamer termed F8-3.1 binds to human FVIII with a K D of 0.67 nM and prolongs aPTT in normal human plasma by 1.9-fold at a concentration of 500 nM. F8-3.1 also has full cross-reactivity to canine FVIII (K D = 0.68 nM) and significantly prolongs the aPTT in canine and porcine plasma. Conclusions : The work describes the generation of aptamers targeting FV/FVa and FVIII that can achieve clinically relevant anticoagulant activity. These results not only demonstrate the feasibility of using cofactor-binding aptamers as therapeutic anticoagulants but also reveal a novel mechanism of aptamer-mediated protein inhibition by interrupting protein-membrane interactions.

Strategies to direct drugs specifically to cancer cells have been increasingly explored, and significant progress has been made toward such targeted therapy. For example, drugs have been conjugated into tumor-targeting antibodies to enable delivery directly to tumor cells. Aptamers are an attractive class of molecules for this type of drug targeting as they are high-affinity/high-specificity ligands, relatively small in size, GMP manufacturable at a large-scale, amenable to chemical conjugation, and not immunogenic. Previous work from our group revealed that an aptamer selected to internalize into human prostate cancer cells, called E3, can also target a broad range of human cancers but not normal control cells. Moreover, this E3 aptamer can deliver highly cytotoxic drugs to cancer cells as Aptamer-highly Toxic Drug Conjugates (ApTDCs) and inhibit tumor growth in vivo. Here, we evaluate its targeting mechanism and report that E3 selectively internalizes into cancer cells utilizing a pathway that involves transferrin receptor 1 (TfR 1). E3 binds to recombinant human TfR 1 with high affinity and competes with transferrin (Tf) for binding to TfR1. In addition, knockdown or knockin of human TfR1 results in a decrease or increase in E3 cell binding. Here, we reported a molecular model of E3 binding to the transferrin receptor that summarizes our findings.

Cell isolation from complex mixtures is a key step in many clinical and research applications, but standard isolation methods may affect the cell's biology and are difficult to reverse. Here, we present a method to isolate and restore cells to their native state using an aptamer that binds epidermal growth factor receptor (EGFR+)cells and a complementary antisense oligonucleotide to reverse binding. For complete details on the use and execution of this protocol, please refer to Gray et al.1