We find that conjugation and chemical composition can alter fundamental aspects of aptamer residence in circulation and distribution to tissues. Though the primary effect of PEGylation was on aptamer clearance, the prolonged systemic exposure afforded by presence of the 20 kDa moiety appeared to facilitate distribution of aptamer to tissues, particularly those of highly perfused organs.

Two molecular sensors that specifically recognize ADP in a background of over 100-fold molar excess of ATP are described. These sensors are nucleic-acid based and comprise a general method for monitoring protein kinase activity. The ADP-aptamer scintillation proximity assay is configured in a single-step, homogeneous format while the allosteric ribozyme (RiboReporter) sensor generates a fluorescent signal upon ADP-dependent ribozyme self-cleavage. Both systems perform well when configured for high-throughput screening and have been used to rediscover a known protein kinase inhibitor in a high-throughput screening format.

Here, we examine biodistribution of radiolabeled aptamers and assess the relative ability of different stabilized aptamer compositions (mixed 2'-F/2'-O-Me; fully 2'-O-Me modified) to access inflamed tissues in a murine inflammation model. Biodistribution of 3H-labeled aptamers, including pegylated and unpegylated compositions, was assessed 3 hours postadministration using quantitative whole body autoradiography (QWBA). Aptamer penetration of cells in kidney and liver was also examined at a qualitative level by microautoradiography. To evaluate aptamer distribution to diseased tissues, inflammation was induced locally in animal hind limbs by treatment with carrageenan just prior to aptamer dosing. Aptamer compositions examined exhibited significant variation in distribution levels among organs and tissues. Highest concentrations of radioactivity in whole body tissues for all animals were observed in the kidney and urinary bladder contents. Relatively little radioactivity was associated with brain, spinal cord, and adipose tissue. Overall, the total level of radioactivity in whole body tissues was significantly higher for a 20-kDa PEG conjugate than for other aptamers. Comparatively high levels of the 20-kDa conjugate were seen in well-perfused organs and tissues, including liver, lungs, spleen, bone marrow, and myocardium. A fully 2'-O-Me composition aptamer had the lowest level of radioactivity in whole body tissues but distributed to higher concentrations in the gastrointestinal tract contents relative to other aptamers. Interestingly, the 20-kDa PEG-conjugated aptamer showed significantly higher levels of distribution to inflamed paw tissues than did either unconjugated or fully 2'-O-Me-modified aptamers.

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Following coronary artery damage, von Willebrand Factor (vWF) multimers adhere to exposed collagen via the vWF A3 domain. Platelets passing through damaged vessels under the conditions of high shear force associated with stenosed arteries will adhere to the vWF A1 domain via GPIb receptors. This initial platelet binding event triggers the formation of a thrombus that can limit blood flow to the myocardium and produce the symptoms of acute coronary syndrome (ACS). Through in vitro selection (SELEX) and subsequent lead optimization, we have generated a nuclease-resistant DNA aptamer that binds to the vWF A1 domain with high affinity. The anti-vWF aptamer inhibits both botrocetin-induced platelet aggregation of platelet rich plasma and shear force-induced platelet aggregation in citrated whole blood. Continuous intravenous infusion of the aptamer in cynomolgus macaques is able to effectively inhibit thrombus formation induced by electrical injury to the carotid artery. Modest effects on template bleeding are observed with the vWF-specific aptamer relative to anti-GPIIb/IIIa therapy. Based upon these results, this aptamer is an excellent candidate for treatment of ACS.