Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer. The crystal structure of thrombin-NU172 complex reveals a bimodular duplex/quadruplex architecture for the aptamer, which binds thrombin exosite I through a highly complementary surface involving all three loops of the G-quadruplex module. Although the duplex domain does not interact directly with thrombin, the features of the duplex/quadruplex junction and the solution data on two newly designed NU172 mutants indicate that the duplex moiety is important for the optimization of the protein-ligand interaction and for the inhibition of the enzyme activity. Our work discloses the structural features determining the inhibition of thrombin by NU172 and put the basis for the design of mutants with improved properties.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

The thrombin-binding aptamer (15-TBA) is a 15-mer DNA oligonucleotide with sequence d(GGTTGGTGTGGTTGG). 15-TBA folds into a quadruplex DNA (G-DNA) structure with two planar G-quartets connected by three single-stranded loops. The arrangement of the 15-TBA-thrombin complex is unclear, particularly with respect to the precise 15-TBA residues that interact with the thrombin structure. Our present understanding suggests either the 15-TBA single stranded loops containing sequential thymidines (TT) or alternatively a single-stranded loop, containing a guanine flanked by 2 thymidines (TGT), physically associates with thrombin protein. In the present study, the explicit solvent molecular dynamics (MD) simulation method was utilized to further analyze the 15-TBA-thrombin three-dimensional structure. Functional annotation of the loop residues was made with long simulations in the parmbsc0 force field. In total, the elapsed time of simulations carried out in this study exceeds 12 microseconds, substantially surpassing previous G-DNA simulation reports. Our simulations suggest that the TGT-loop function is to stabilize the structure of the aptamer, while the TT-loops participate in direct binding to thrombin. The findings of the present report advance our understanding of the molecular structure of the 15-TBA-thrombin structure further enabling the construction of biosensors for aptamer bases and the development of anticoagulant agents.

Characteristics of a new antithrombin DNA-aptamer RE31 were studied. This aptamer inhibited thrombin formation in human plasma catalyzed by exogenous (lengthening of thrombin time) and endogenous thrombin (lengthening of partial prothrombin time and activated partial thromboplastin time). In addition, the aptamer completely suppressed thrombin-induced aggregation of human platelets. On the other hand, RE31 did not reduce amidolytic activity of thrombin towards the short peptide substrate, in other words, did not modify the state of enzyme active center. By the capacity to inhibit clotting reactions, RE31 was superior to the previously described highly effective 31-component antithrombin aptamer 31TBA (thrombin binding aptamer, TBA). The effect of RE31 was species-specific: it inhibited human thrombin activity more effectively than activities of rat and rabbit thrombins.

A thermostable cyclodextrin glucanotransferase (CGTase) was isolated from a Bacillus stearothermophilus strain, ET1, which was screened from Korean soil. The corresponding CGTase gene cloned in Escherichia coli shared 84% and 88% identity with CGTase genes from other B. stearothermophilus strains at the nucleotide and amino acid sequence level, respectively. The enzyme was purified to apparent homogeneity by β-cyclodextrin (CD) affinity chromatography and high-performance liquid chromatography. The enzyme had an apparent molecular mass of 66,800 Da and a pI of 5.0. The optimum pH for the enzyme-catalyzed reaction was pH 6.0, and the optimum temperature was observed at 80 °C. Thermostability of the enzyme was enhanced by Ca2+. A 13% (w/v) cornstarch solution was liquefied and converted to CDs solely using this enzyme. The cornstarch conversion rate was 44% and α-, β-, and γ-CDs were produced in the ratio of 4.2:5.9:1. Keywords: Cyclodextrin glucanotransferase; thermostability; cyclodextrin; Bacillus stearothermophilus

Edited by Judit OvádiKeywords: Aptamer Thrombin Prothrombin Surface plasmon resonance Equilibrium constant Kinetic constant a b s t r a c t Structural properties determine binding affinities of DNA aptamers specific to thrombin. Our paper is the first to focus on a family of eight G-quadruplex-based aptamers with varied duplex region length (from two to eight base pairs). We have shown that the duplex, which is not the main binding domain, greatly influences the interaction with thrombin and prothrombin. Furthermore, the affinity of an aptamer to thrombin and prothrombin increases (respectively from 2.7 Â 10 À8 M to 5.6 Â 10 À10 M and from 1.8 Â 10 À5 M to 7.1 Â 10 À9 M) with an increase in the number of nucleotide pairs in the duplex region.

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.

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