The tertiary structure of a DNA aptamer which binds to and inhibits thrombin determines activity

Wang, Ke Yu; Krawczyk, Steven H.; Bischofberger, Norbert; Swaminathan, S.; Bolton, Philip H.

doi:10.1021/bi00093a004

Cited by 176 publications

(165 citation statements)

References 25 publications

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“…The predominance of the G-aptamer in solution is attributed to the stabilizing effect of the metal ions that have the right size (1.3∼1.5 Å) to fit into the quadruplex center and coordinate with the two G-quartets [28,38]. This tertiary Gquadruplex structure is essential for the aptamer to bind thrombin molecules [39,40]. Among all the cations, K + is most commonly used due to its optimal effect on the Gquadruplex and its propensity to have little interaction with the inner surfaces of the capillary or microchip channels.…”

Section: Cation Effectmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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The use of traditional capillary electrophoresis (CE) to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombinbinding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with a limit of detection for thrombin of 5 nM at a 3-fold signal-to-noise ratio.

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Section: Cation Effectmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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“…The formation of nucleic acids secondary structures, such as hairpin loops, triplexes, G-quadruplexes, pseudoknots, and i-motifs is well documented [1][2][3][4] and they have been postulated to be involved in a variety of biological functions [4][5][6][7][8][9][10][11][12][13][14]. Pseudoknots belong to an interesting and diverse RNA structural motif, due to variation in their loop lengths and stems and the types of interactions between them.…”

Section: Introductionmentioning

confidence: 99%

Contributions of the loops on the stability and targeting of DNA pseudoknots

Reiling¹,

Marky²

2014

Bio Chem Comp

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Background: Pseudoknots have been found to play important roles in RNA function, examples include ribosomal frameshifting and in the 5' UTR of mRNA as riboswitches. In RNA frameshifting, there is a local formation of base-triplet stacks within the pseudoknot, increasing the stability of the terminal stem. The interaction of this triplex structure with the ribosome might help with the high-efficiency of frameshifting. The main objective of this work is to mimic RNA pseudoknots using DNA oligonucleotides for the control of gene expression. Specifically, we have designed a pair of DNA pseudoknots with different length in one of the loops to mimic the formation of a local triple helix, shown within RNA pseudoknots of the human telomerase. Methods: We have used a combination of temperature-dependent UV spectroscopy and calorimetric techniques to determine the thermodynamics for the unfolding of the pseudoknots and their targeting with complementary strands. The unfolding data is then used to create thermodynamic (Hess) cycles that correspond to each of the targeting reactions. The resulting data is then compared with the thermodynamic enthalpy data obtained directly from isothermal titration calorimetry. Results: UV melting curves of each pseudoknot show transitions with T M s independent of strand concentration, which confirms their intramolecular formation. Analysis of the differential scanning calorimetry (DSC) curves shows the pseudoknot with the longer thymine loop (PsK-9) to be more stable, by -5.7 kcal/mol, and to unfold with a higher enthalpy of 27.4 kcal/mol. The targeting of each pseudoknot yielded favorable reaction free energy contributions that were enthalpy driven. However, the disruption reaction of PsK-9 took place with a less favorable free energy term, by 0.7 kcal/mol, and less favorable enthalpy term, by 4.5 kcal/mol. Conclusion: The thermodynamic unfolding data showed that PsK-9 is more stable or more compact, due to the involvement of three loop thymines of PsK-9 in forming three T*AT base-triplets, or two T*AT/ T*AT base-triplet stacks, in the stem of this pseudoknot. The targeting thermodynamic data indicated that each complementary strand is able to disrupt the pseudoknots. However, the disruption of PsK-9 takes place with a less favorable free energy contribution, confirming the formation of a short and local triplex.

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“…There are two anti-thrombin aptamers with high affinity. One is a 15-mer single-stranded DNA which forms an intramolecular quadruplex structure and binds to thrombin at the fibrinogen-recognition exosite [26,27] been proved to bind at heparin-binding exosite with a higher affinity [28]. When these aptamers are used in APCE/LIF, the assay has showed potential properties of smaller sample size, high sensitivity, rapid separation, and compatibility with automation [29] for thrombin detection.…”

Section: Introductionmentioning

confidence: 99%

Highly sensitive detection of human thrombin in serum by affinity capillary electrophoresis/laser-induced fluorescence polarization using aptamers as probes

Song

Zhang

et al. 2009

Journal of Chromatography A

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a b s t r a c tThe detection and quantification of disease-related proteins play critical roles in clinical practice and diagnostic assays. We present an affinity probe capillary electrophoresis/laser-induced fluorescence polarization (APCE/LIFP) assay for detection of human thrombin using a specific aptamer as probe. In the APCE/LIFP assay, the mobility and fluorescence polarization of complex are measured simultaneously during CE analysis. The affinity complex of human thrombin can be well separated from unbound aptamer on CE and clearly identified on the basis of its fluorescence polarization and migration. Because of the binding favorable G-quartet conformation potentially involved in the specific aptamer, it was assumed that monovalent and bivalent cations promoting the formation of a stable G quadruplex conformation in the aptamer may enhance the binding of the aptamer and thrombin. Therefore, we investigated the effects of various metal cations on the binding of human thrombin and the aptamer. Our results show that cations like K + and Mg 2+ could not stabilize the affinity complex. Without the use of typical cations, a highly sensitive assay of human thrombin was developed with the corresponding detection limits of 4.38 × 10 −19 and 2.94 × 10 −19 mol in mass for standard solution and human serum, respectively.

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The tertiary structure of a DNA aptamer which binds to and inhibits thrombin determines activity

Cited by 176 publications

References 25 publications

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Contributions of the loops on the stability and targeting of DNA pseudoknots

Highly sensitive detection of human thrombin in serum by affinity capillary electrophoresis/laser-induced fluorescence polarization using aptamers as probes

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