The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer.

Padmanabhan, K.; Ferrara, Joseph D.; Sadler, J. Evan; Tulinsky, A.

doi:10.1016/s0021-9258(17)46749-4

Cited by 450 publications

(183 citation statements)

References 29 publications

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“…Within very few years from the initial structural characterization of thrombin, structures of complexes with rather large partners were determined. These studies unraveled the interaction modes of this protein with the kringle-2 domain of prothrombin [ 43 ]; with functional partners (thrombomodulin [ 114 ], heparin [ 115 , 116 ], platelet glycoprotein Ibα [ 117 , 118 ], and fibrinogen [ 119 , 120 ]); with the natural inhibitors rhodniin [ 72 ], ornithodorin [ 70 ], triabin [ 30 ], hemadin [ 61 ], staphylocoagulase [ 73 ]; and with aptamers (initially TBA [ 121 ] and, then, Toggle-25t [ 87 ]) ( Table 2 and Table 3 ). In particular, TBA is the first DNA aptamer selected against thrombin [ 122 ].…”

Section: Structural Characterization Of Thrombin: a Chronological Perspectivementioning

confidence: 99%

“…In particular, TBA is the first DNA aptamer selected against thrombin [ 122 ]. It adopts an antiparallel G-quadruplex structure and recognizes the exosite I [ 80 , 121 ]. Conversely, Toggle-25t is an RNA aptamer that contains 2′-fluoropyrimidine nucleotides and binds thrombin exosite II [ 87 ].…”

Section: Structural Characterization Of Thrombin: a Chronological Perspectivementioning

confidence: 99%

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Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

Troisi

Balasco

Autiero

et al. 2021

IJMS

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Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.

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Section: Structural Characterization Of Thrombin: a Chronological Perspectivementioning

confidence: 99%

Section: Structural Characterization Of Thrombin: a Chronological Perspectivementioning

confidence: 99%

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

Troisi

Balasco

Autiero

et al. 2021

IJMS

View full text Add to dashboard Cite

show abstract

“…It is usually stabilized by a metal cation (Na + or K + ) in between the two quartets [11]. TBA binds to thrombin exosite I via the T-T loops and exosite II is a heparin binding site for the T-G-T loop [12][13][14][15]. Currently, one of the TBA variants is in clinical trials as an anticoagulant for patients undergoing coronary artery bypass graft surgery [16].…”

Section: Introductionmentioning

confidence: 99%

Structural and Binding Effects of Chemical Modifications on Thrombin Binding Aptamer (TBA)

et al. 2021

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The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5′ end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (KD) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA–thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.

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“…This E spacer was incorporated at single or double positions in the T 3 T 4 and T 7 G 8 T 9 loops of TBA, as the TT and TGT loops are implicated in the interaction with, and subsequent inhibition of, thrombin. [13,14] The abasic ethylene glycol spacer phosphoramidite was synthesized employing simple chemical transformations as outlined in Scheme 1 and slight modification of earlier reported procedures. [15] Accordingly, commercially available ethylene glycol was converted to the mono-dimethoxytrityl (DMTr)- protected compound 2 on treatment with DMTr-chloride in presence of 4-dimethylaminopyridine (DMAP) with pyridine as a solvent in 60 % yield.…”

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confidence: 99%

Replacement of Loop Residues in TBA by an Abasic Ethylene Glycol Spacer: Effect on Stability, Structure and Function**

2021

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This article describes the synthesis of ethyleneglycol (E) phosphoramidite and its incorporation into the thrombin binding aptamer (TBA) sequence at loop positions. Circular dichroism (CD) study revealed no major disturbances in the secondary structure of TBA by the abasic E unit and the derived oligomers exhibited a typical antiparallel chair‐like conformation similar to that of TBA. UV and CD spectroscopy, together with anti‐coagulation and HPLC studies revealed that although nuclease stability was enhanced, and anti‐coagulation reasonably good, the thermal stability of the quadruplexes was adversely affected.

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The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer.

Cited by 450 publications

References 29 publications

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

Structural and Binding Effects of Chemical Modifications on Thrombin Binding Aptamer (TBA)

Replacement of Loop Residues in TBA by an Abasic Ethylene Glycol Spacer: Effect on Stability, Structure and Function**

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