e c h n i c a l r e p o r t sWith an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that proteinand polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics.

Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68-and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steadystate kinetic parameters for both aminoacyl-␤-naphthylamide and unmodified dipeptide substrates over the pH range 5.0 -8.5 reveals that k cat is relatively insensitive to pH, whereas K m increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0 -5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.Human malaria is responsible for around one million deaths annually (1). Five species of the genus Plasmodium cause malaria in humans as they replicate within host erythrocytes. The cytoadherent properties of intraerythrocytic Plasmodium falciparum coupled with its ability to invade mature erythrocytes make it the most virulent of the species that infect humans. During its erythrocytic replication cycle, P. falciparum endocytoses and catabolizes over two-thirds of soluble erythrocyte proteins (2, 3), the majority of which is hemoglobin. Hemoglobin catabolism provides amino acids for protein synthesis, general metabolism, and isoleucine import (4, 5) and may also prevent premature hemolysis by reducing the colloid osmolarity of the erythrocyte (6). Blocking hemoglobin catabolism with protease inhibitors prevents parasite replication; therefore, enzymes that catalyze this process are attractive targets for the development of novel anti-malarial drugs (7).Hemoglobin is extensively catabolized by the parasite within an acidic organelle called the food vacuole or digestive vacuole. In the vacuole, numerous types of endo-and exopeptidases act in a complementary and concerted manner to catalyze the hydrolysis of the ␣-and ␤-globin chains of hemoglobin. Aspartic proteases (plasmepsin I, II, IV, and histo-aspartic protease) and cysteine endoproteases (falcipain-2, -2Ј, and -3) initiate cleavage of the globin chains and generate polypeptide fragments (8, 9). The metallopeptidase fa...

Background The conversion of prothrombin to thrombin is one of two non-duplicated enzymatic reactions during coagulation. Thrombin has long been considered an optimal anticoagulant target because it plays a crucial role in fibrin clot formation by catalyzing the cleavage of fibrinogen, upstream coagulation cofactors, and platelet receptors. Although a number of anti-thrombin therapeutics exist, it is challenging to use them clinically due to their propensity to induce bleeding. Previously, we isolated a modified RNA aptamer (R9D-14) that binds prothrombin with high affinity and is a potent anticoagulant in vitro. Objectives We sought to explore the structure of R9D-14 and elucidate its anticoagulant mechanism(s). In addition to designing an optimized aptamer (RNAR9D-14T), we also explored whether complementary antidote oligonucleotides can rapidly modulate the optimized aptamer’s anticoagulant activity. Methods and Results RNAR9D-14T binds prothrombin and thrombin pro/exosite I with high affinity and inhibits both thrombin generation and thrombin exosite I-mediated activity (i.e., fibrin clot formation, feedback activity, and platelet activation). RNAR9D-14T significantly prolongs the aPTT, PT, and TCT clotting assays, and is a more potent inhibitor than the thrombin exosite I DNA aptamer ARC-183. Moreover, a complementary oligonucleotide antidote can rapidly (<2 min) and durably (>2 hrs) reverse RNAR9D-14T anticoagulation in vitro. Conclusions Powerful anticoagulation, in conjunction with antidote reversibility suggests that RNAR9D-14T may be ideal for clinical anticoagulation in settings that require rapid and robust anticoagulation, such as cardiopulmonary bypass, deep vein thrombosis, stroke, or percutaneous coronary intervention.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function via unique, previously unappreciated mechanisms. Here, utilizing a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.