Anti-thrombin aptamers

The first anti-thrombin aptamer, TBA, was generated through via SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology in 1992 by L.C.

[1] A second thrombin-binding aptamer, HD22, recognizes thrombin exosite II and was discovered in 1997 by NeXstar (now Gilead Sciences).

[2] These two aptamers have high affinity and good specificity and have been widely studied and used for the development of aptamer-based therapeutics and diagnostics.

The dissociation constant of TBA-thrombin has been reported in nano-molar range, and TBA does not interact with other plasma proteins or thrombin analogues (e.g., gamma-thrombin).

[3] As a result, TBA has been used as a short-term anti-coagulant designed for the application in the coronary artery bypass graft surgery, and its optimized form (NU172) is now under the phase II of clinical trial by ARCA Biopharma (NCT00808964).

[4] Also, due to its high affinity and specificity, a variety of sensors was coupled with TBA and developed for thrombosis diagnostics.

[8] The melting temperature of TBA's G-quadruplex (measuring the intensity change of the peak at 295 nm by CD) in the presence of sodium ion and potassium are 24 °C and 53 °C, respectively.

In contrast, due to its small size, sodium ion can only interacts with four rather than eight oxygen atoms of two G-tetrad planes, and accordingly has two alternative position in the cavity.

In the ion-deficient condition, thrombin helps TBA form into a stable G-quadruplex structure from a randomized coil, which results in conformational change.

For this purpose, TBA is usually mounted with an additional sequence with a FRET (Förster resonance energy transfer) pair to form a transient duplex structure.

[10] TBA is bound to the exosite I of thrombin majorly via its two TT loops (T3, T4 and T12, T13) through polar and hydrophobic interactions.

However, in the presence of sodium ion, the hydrogen bonding between T3 and His71 is lost, and the intermolecular distance is longer than that in the potassium case.

[16] TBA entered the phase I clinical trial for coronary artery bypass graft surgery by Archemix and Nuvelo (now ARCA Biopharma) around 2005.

[17] Thus, the companies redesigned the sequence of TBA and developed a second-generation 26-mer DNA aptamer known as NU172, which is now under phase II clinical trial.

Since the exosite II is a positively charged motif, it creates many ion pairs with the HD22 backbone especially in the duplex region.

Moreover, Interacting with thrombin improves the thermal stability of HD22 structure, and results in the increase of melting temperature (from 36 to 48 °C).

The complexes of (A) TBA-thrombin and (B) HD22-thrombin (PDB files 4DII and 4I7Y). The protein and aptamer were represented in the ribbon and ball&stick formats, respectively.
The G-quadruplex structure adopted by TBA. (A) The crystallographic structure and (B) the schematic illustration of TBA (PDB file 4DII). Insert: the top layer of G-tetrad (The Hoogsteen-like hydrogen bonds are highlighted with green dashed lines).
The interactions between TBA and ions. (A) TBA-potassium ion complex. Potassium ion fits the cavity between the two G-tetrad planes of TBA properly and coordinately interacts with eight O6 atoms in G-quadruplex. (insert: the whole structure of TBA-K+ complex) (B) TBA-sodium ion complex. Two alternative positions of sodium observed, and sodium can only interacts with four rather than eight oxygens.
The interface between TBA and the exosite I of thrombin. (A) The interface. Involved protein residues and aptamer nucleotides are labeled with red and green, respectively. (B) The interaction between His71 and T3 (TBA) in the presence of potassium ion. (C) The positions of His 71 and T3 (TBA) in the presence of sodium ion. (D) The positions of His71 and T3 (mTBA). Dots represent the interactions between thrombin and aptamer.
HD22 structure and interbase interaction. (A) Overall structure of HD22. (B) Top G-tetrad plane (C) The Watson-Crick base pairs in the G-quadruplex motif. (D) G-fork interaction between G-quadruplex and duplex motifs
HD22-exosite II interaction. (A) Overall interface between HD22 and the exosite II. (B) The interface at the duplex motif. (C) The interface at the G-quadruplex motif. Dots represent the interactions.