Rapid Complexation of Aptamers by Their Specific Antidotes

Abstract

Nucleic acid ligands, aptamers, harbor the unique characteristics of small molecules and antibodies. The specificity and high affinity of aptamers enable their binding to different targets, such as small molecules, proteins, or cells. Chemical modifications of aptamers allow increased bioavailability. A further great benefit of aptamers is the antidote (AD)-mediated controllability of their effect. In this study, the AD-mediated complexation and neutralization of the thrombin binding aptamer NU172 and Toll-like receptor 9 (TLR9) binding R10-60 aptamer were determined. Thereby, the required time for the generation of aptamer/AD-complexes was analyzed at 37 °C in human serum using gel electrophoresis. Afterwards, the blocking of aptamers’ effects was analyzed by determining the activated clotting time (ACT) in the case of the NU172 aptamer, or the expression of immune activation related genes IFN-1β, IL-6, CXCL-10, and IL-1β in the case of the R10-60 aptamer. Gel electrophoresis analyses demonstrated the rapid complexation of the NU172 and R10-60 aptamers by complementary AD binding after just 2 min of incubation in human serum. A rapid neutralization of anticoagulant activity of NU172 was also demonstrated in fresh human whole blood 5 min after addition of AD. Furthermore, the TLR9-mediated activation of PMDC05 cells was interrupted after the addition of the R10-60 AD. Using these two different aptamers, the rapid antagonizability of the aptamers was demonstrated in different environments; whole blood containing numerous proteins, cells, and different small molecules, serum, or cell culture media. Thus, nucleic acid ADs are promising molecules, which offer several possibilities for different in vivo applications, such as antagonizing aptamer-based drugs, immobilization, or delivery of oligonucleotides to defined locations.

Application of Piezo-Based Measuring System for Evaluation of Nucleic Acid-Based Drugs Influencing the Coagulation

“…Furthermore, using a ball coagulometer, slightly higher CTs were also detected in blood samples with 1.0 µM (NU172+ AD) (both time points) than in the control (NaCl), which indicates a remaining amount of NU172 that was not complexed with AD. In accordance with our previous publication [7], already after 5 min of incubation with the AD, the anticoagulation effect of NU172 was abolished and maintained during the dynamic incubation in the in vitro rotation model of 30 min. Figure 7.…”

“…In this study, using the new PIEZ system, we were able to reliably measure the coagulation inhibiting effect of the thrombin binding aptamer NU172 after static and dynamic incubation and the ability of its complementary antidote (AD) to abrogate the coagulation inhibitory effect of NU172. In our previous study, the required time for the generation of double-stranded complexes was examined [7], and it was found that the AD was able to bind already after 2 min to the NU172 aptamer, which resulted in rapid abrogation of the anticoagulant activity of NU172 after 5 min in whole blood.…”

“…The incubation of blood was performed with the thrombin binding aptamer (NU172) and its complementary single-stranded (ss) DNA (AD) ( Table 1). Furthermore, a nonsense aptamer (NS) and a nonsense antidote (NS_AD) were used as negative controls [7]. All oligonucleotides were ordered HPLC-purified from Ella Biotech GmbH (Martinsried, Germany).…”

“…The DNA aptamer NU172 is selected against thrombin and specifically binds to the anionic binding site exosite I of thrombin, which has two exosites, exosite I and II. As a result, fibrinogen cannot be transformed into fibrin and the coagulation process is inhibited [5,7]. Thus, coagulation inhibiting aptamers, such as thrombin binding aptamer NU172, represent a promising alternative to the use of heparin in HIT patients during ECC.…”

Comparison of Effects of Anti-thrombin Aptamers HD1 and HD22 on Aggregation of Human Platelets, Thrombin Generation, Fibrin Formation, and Thrombus Formation Under Flow Conditions

“…The conjugation of polyethylene glycol (PEG) to aptamers delay their renal filtration (Padilla and Sousa, 1999; Sun et al, 2014; Ni et al, 2017; Maio et al, 2018). The pharmacokinetic parameters of an aptamer could be also controlled by an antidote being a complementary oligonucleotide (Oney et al, 2010; Lakhin et al, 2013; Stoll et al, 2017).…”