Investigation of the selectivity of thrombin-binding aptamers for thrombin titration in murine plasma

Abstract

Detection of thrombin in plasma raises timely challenges to enable therapeutic management of thrombosis in patients under vital threat. Thrombin binding aptamers represent promising candidates as sensing elements for the development of real-time thrombin biosensors; however implementation of such biosensor requires the clear understanding of thrombin-aptamer interaction properties in real-like environment. In this study, we used Surface Plasmon Resonance technique to answer the questions of specificity and sensitivity of thrombin detection by the thrombin-binding aptamers HD1, NU172 and HD22. We systematically characterized their properties in the presence of thrombin, as well as interfering molecular species such as the thrombin precursor prothrombin, thrombin in complex with some of its natural inhibitors, nonspecific serum proteins, and diluted plasma. Kinetic experiments show the multiple binding modes of HD1 and NU172, which both interact with multiple sites of thrombin with low nanomolar affinities and show little specificity of interaction for prothrombin vs. thrombin. HD22, on the other hand, binds specifically to thrombin exosite II and has no affinity to prothrombin at all. While thrombin in complex with some of its inhibitors could not be recognized by any aptamer, the binding of HD1 and NU172 properties is compromised by thrombin inhibitors alone, as well as with serum albumin. Finally, the complex nature of plasma was overwhelming for HD1, but we define conditions for the thrombin detection at 10nM range in 100-fold diluted plasma by HD22. Consequently HD22 showed key advantage over HD1 and NU172, and appears as the only alternative to design an aptasensor.

Self-bending symmetric cusp beams

“…Thrombin detection in murine plasma using engineered fluorescence resonance energy transfer aptadimers Ana Trapaidze, 1,2 Marie Brut, 1,2 Serge Mazè res, 3 Daniel Estève, 1,2 Anne-Marie Gu e, 1,2 and Aur elien Bancaud 1,2,a) 1 CNRS, LAAS, 7 avenue du colonel Roche, F-31400 Toulouse, France Biodetection strategies, in which two sides of one target protein are targeted simultaneously, have been shown to increase specificity, selectivity, and affinity, and it has been suggested that they constitute excellent candidates for protein sensing in complex media. In this study we propose a method to engineer the sequence of a DNA construct dedicated to reversible thrombin detection.…”

“…A number of specific aptamers, such as HD1, NU172, or HD22, have been used in the literature, 1 yet we recently showed that the poor selectivity of HD1 or NU172 was incompatible with the detection of thrombin in plasma, whereas HD22 seemed to be more selective, allowing the detection of thrombin in diluted murine plasma. 2 Nevertheless, the high nonspecific background encountered in complex media can be efficiently diminished by changing the detection format with double-site interaction, in particular, using sandwich assays. [3][4][5][6][7][8][9][10][11][12][13] Alternatively, the proximity between two aptamers that bind different thrombin epitopes has been exploited to collect a specific signal of the target.…”

“…We note that this result was consistent with our observation that thrombin could be detected by Surface Plasmon Resonance (SPR) with HD22 in 100-fold, but not in 10-fold diluted plasma. 2 Altogether we presented a method to design FRET aptadimers for recognition and quantification of thrombin. Our approach is integrated, involving (i) the development of software to define the linker sequence according to rules focused on the preservation of aptamer structure and stability, and (ii) the confirmation of aptadimer folding and functionality with FRET-based scanning temperature fluorimetry.…”

Thrombin detection in murine plasma using engineered fluorescence resonance energy transfer aptadimers

“…Given the low selectivity of HD1 in comparison with HD22, 2 we wished to clarify whether the global sensing properties of the construct were sufficient to discriminate thrombin from its precursor prothrombin, which is abundantly present in serum. Temperature scanning fluorimetry of the aptadimer alone or in the presence of an equimolar concentration of prothrombin (Stago, France) yielded similar responses with marginal changes in intensity at 30 C (Fig.…”

“…A number of specific aptamers, such as HD1, NU172, or HD22, have been used in the literature, 1 yet we recently showed that the poor selectivity of HD1 or NU172 was incompatible with the detection of thrombin in plasma, whereas HD22 seemed to be more selective, allowing the detection of thrombin in diluted murine plasma. 2 Nevertheless, the high nonspecific background encountered in complex media can be efficiently diminished by changing the detection format with double-site interaction, in particular, using sandwich assays. [3][4][5][6][7][8][9][10][11][12][13] Alternatively, the proximity between two aptamers that bind different thrombin epitopes has been exploited to collect a specific signal of the target.…”