Gold Nanoparticles Presenting Hybridized Self-Assembled Aptamers That Exhibit Enhanced Inhibition of Thrombin
Die DNA-Hybridisierung Thrombin-bindender Aptamere (TBAs) unter Bildung einer selbstorganisierten Monoschicht von Goldnanopartikeln (Au-NPs) wurde genutzt, um die Koagulationswirkung von Thrombin zu steuern. Die hTBA15/hTBA29/cDNA-Au-NPs konnten die hTBA15- und hTBA29-Einheiten in geeignetem Abstand und passender Orientierung binden, sodass sie stark mit Thrombin wechselwirken und die Koagulation extrem hemmen können.
[Show abstract] [Hide abstract] ABSTRACT: Nucleic acid-based aptamers can be selected from combinatorial libraries of synthetic oligonucleotides to bind, with affinity and specificity similar to antibodies, a wide range of biomedically relevant targets. Compared to protein therapeutics, aptamers exhibit significant advantages in terms of size, non-immunogenicity and wide synthetic accessibility. Various chemical modifications have been introduced in the natural oligonucleotide backbone of aptamers in order to increase their half-life, as well as their pharmacological properties. Very effective alternative approaches, devised in order to improve both the aptamer activity and stability, were based on the design of polyvalent aptamers, able to establish multivalent interactions with the target: thus, multiple copies of an aptamer can be assembled on the same molecular- or nanomaterial-based scaffold. In the present review, the thrombin binding aptamers (TBAs) are analyzed as a model system to study multiple-aptamer constructs aimed at improving their anticoagulation activity in terms of binding to the target and stability to enzymatic degradation. Indeed - even if the large number of chemically modified TBAs investigated in the last 20years has led to encouraging results - a significant progress has been obtained only recently with bivalent or engineered dendritic TBA aptamers, or assemblies of TBAs on nanoparticles and DNA nanostructures. Furthermore, the modulation of the aptamers activity by means of tailored drug-active reversal agents, especially in the field of anticoagulant aptamers, as well as the reversibility of the TBA activity through the use of antidotes, such as porphyrins, complementary oligonucleotides or of external stimuli, are discussed.0Comments 31Citations
- "8. Schematic representation of the binding and thrombin activity inhibition of TBA 15 and TBA 29 units conjugated to gold nanoparticles through Watson–Crick H-bonds. Adapted and modified from Shiang et al. (2011). TBA binding site, a caged thymidine inserted in a key location (T4) is sufficient to completely mask the aptamer functions, i.e. its affinity for thrombin and inhibition of the blood clotting cascade (Heckel & Mayer, 2005). "
- [Show abstract] [Hide abstract] ABSTRACT: In this paper, we have demonstrated that the thymine linker length number (Tn, n = 0–60) and stem pair number (Pm, m = 0–16) in the terminal of thrombin binding aptamers (TBAs) have a strong impact on the flexibility and stability of TBA-modified gold nanoparticles (TBA–Au NPs) and thus the binding strength and inhibitory potency toward thrombin. The anticoagulation of TBA–Au NPs increased with an increase in the linker length from T0 to T30 due to an increase in the flexibility of G-quadruplexes of TBAs on the Au NP surfaces (TBA-Tn–Au NPs). The inhibition of TBA-PmT15–Au NPs increased with an increase in the Pm from P0 to P8 as a result of the increase in the rigidity and the stability of G-quadruplexes of the TBAs on the Au NP surfaces. The best results were observed for multivalent TBA–Au NPs conjugates—TBA15/TBA29-P8T15–Au NPs—which exhibited ultra-high binding affinity toward thrombin (Kd = 8.86 × 10−12 M) and thus extremely high anticoagulant (inhibitory) potency because of their particularly flexible and stable conformation and multivalency. Compared to the case without inhibitors, their measured thrombin clotting time (TCT) was 296 times longer, whereas for TBA15 alone it was only 3.9 times longer. From the dosage dependence of the TCT delay, we further demonstrated the anticoagulation ability of our TBA15/TBA29-P8T15–Au NPs was much better than the commercial drugs (argatroban and hirudin). Moreover, the Au NPs modified with TBA with photocleavable (PC) units allow a reversal in the activity of TBAPC–Au NPs via near-UV light-inducement of TBA release from Au NPs. We believe that our described techniques can be used widely to modify NPs with other anticoagulantDNA or RNA aptamers towards different proteins such as factor IX, activated protein C, and factor VIIa.0Comments 7Citations
- [Show abstract] [Hide abstract] ABSTRACT: We prepared thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs) through a molecularly imprinted (MP) approach, which provide highly efficient inhibition activity toward the polymerization of fibrinogen. Au NPs (diameter, 13 nm), 15-mer thrombin-binding aptamer (TBA(15)) with different thymidine linkers, and 29-mer thrombin-binding aptamer (TBA(29)) with different thymidine linkers (Tn) in the presence of thrombin (Thr) as a template were used to prepare MP-Thr-TBA(15)/TBA(29)-Tn-Au NPs. Thrombin molecules were then removed from Au NPs surfaces by treating with 100 mM Tris-NaOH (pH ca. 13.0) to form MP-TBA(15)/TBA(29)-Tn-Au NPs. The length of the thymidine linkers and TBA density on Au NPs surfaces have strong impact on the orientation, flexibility, and stability of MP-TBA(15)/TBA(29)-Tn-Au NPs, leading to their stronger binding strength with thrombin. MP-TBA(15)/TBA(29)-T(15)-Au NPs (ca. 42 TBA(15) and 42 TBA(29) molecules per Au NP; 15-mer thymidine on aptamer terminal) provided the highest binding affinity toward thrombin with a dissociation constant of 5.2 × 10(-11) M. As a result, they had 8 times higher anticoagulant (inhibitory) potency relative to TBA(15)/TBA(29)-T(15)-Au NPs (prepared in the absence of thrombin). We further conducted thrombin clotting time (TCT) measurements in plasma samples and found that MP-TBA(15)/TBA(29)-T(15)-Au NPs had greater anticoagulation activity relative to four commercial drugs (heparin, argatroban, hirudin, and warfarin). In addition, we demonstrated that thrombin induced the formation of aggregates from MP-TBA(15)-T(15)-Au NPs and MP-TBA(29)-T(15)-Au NPs, thereby allowing the colorimetric detection of thrombin at the nanomolar level in serum samples. Our result demonstrates that our simple molecularly imprinted approach can be applied for preparing various functional nanomaterials to control enzyme activity and targeting important proteins.0Comments 18Citations