Light-controlled thrombin catalysis and clot formation using a photoswitchable G-quadruplex DNA aptamer

Abstract

The reversible photocontrol of an enzyme governing blood coagulation is demonstrated. The thrombin binding aptamer (TBA), was rendered photochromic by modification with two anthracene groups. Lighttriggered anthracene photodimerisation distorts its structure, inhibiting binding of the enzyme thrombin, which in turn triggers catalysis and the resulting clotting process.External control of biomolecule function is important for regulating biological processes that are relevant to various therapeutic and diagnostic applications. Photoregulation is a popular method of control due to the high level of specificity gained without the use of additional chemical reagents, 1 with photoswitchable groups allowing properties to be controlled in a reversible manner. As part of ongoing interest in this area, there are now a number of examples of different photoactive groups used for photocontrollable catalysis. 1a,2,3 Here, through the use of a photoswitchable DNA aptamer that displays anthracene photochromism, we demonstrate a new way of using light to reversibly control a catalytic process.Human a-thrombin, an enzyme from the family of serine proteases, is a key biological protein involved in the process of blood coagulation at the site of a blood vessel injury. 4 It has two anion binding exosites, one of which (exosite I) binds fibrinogen. During the formation of a blood clot, thrombin catalyses the production of insoluble fibrin polymers from soluble fibrinogen monomers. DNA aptamers, short single stranded oligonucleotides generated synthetically to bind proteins and small molecules, are widely used for a range of applications linked to diagnostics and therapy. 5 The thrombin binding aptamer or TBA (Fig. 1) is a 15-mer that adopts an anti-parallel G-quadruplex (G4) tertiary structure in the presence of K + ions and binds to thrombin at exosite I, the fibrinogen binding site. 6,7 The resulting complex competitively inhibits the binding of the enzyme to fibrinogen, thereby inhibiting the process of blood coagulation. This has led to the consideration of TBA and related compounds as treatments for medical conditions linked to blood clotting disorders. 5d However the use of anticoagulants, including traditional ones (e.g. heparin or warfarin), can often result in other healthcare risks. 8 For example, the reduction in systemic clotting capability could mean that wound healing is compromised at injury sites away from where anticoagulation is required. A dynamic system in which anticoagulation could be deactivated locally using an orthogonal and external source, and more quickly than the body's clearance systems, could provide an effective solution to such an issue.Previous examples exploring this idea using TBA derivatives 2d, f,7b have included azobenzene 2d and nitrophenyl 2f systems in which the application of light was found to alter clotting times. Anthracene photochromism is known to be particularly effective in photoregulatory processes within supramolecular systems as its reversible photodimerisation reaction can induce s...

Photoresponsive Control of G-Quadruplex DNA Systems

“…More recently, the Tucker group engineered a bisanthracene-functionalized G4 thrombin-binding aptamer, whereby (4π + 4π) photocycloaddition reaction of two anthracene groups can modulate the binding, and therefore activity, of thrombin ( Figure 1 c). 67 The group hypothesize the observed control of bioactivity can be attributed to the distortion of the structure, which inhibits the binding to thrombin. Unlike, the previous examples, the reversible photodimerization of the anthracene unit occurs using both light and thermal triggers, which limits somewhat their potential applications in a biological setting.…”

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

“…To overcome this problem while increasing the anti‐clotting effect, many nucleotide and backbone modifications have been reported [6] . Among the studies that involved replacement of the loop residues by abasic units, were those that introduced a dibenzyl linker, [7] anthraquinone units, [8] tetrapeptides, [9] a 3‐carbon abasic spacer, [10] anthracene groups, [11] and azobenzene derivatives [12] . An enhancement in the thermal stability of the resulting quadruplexes was reported for the dibenzyl linker, [7] double anthraquinone units, [8] single anthracene substitution at T9, [11] C3‐spacer [10] and selected azobenzene substitutions [12] .…”

Acrylamide-dT: a polymerisable nucleoside for DNA incorporation

“…Therefore these conditions were chosen for automated oligonucleotide synthesis. Given the ongoing interest in functionalising aptameric DNA sequences with polymerisable groups, 9,10,17,18 and our recent work on such systems, 21 it was decided to incorporate Acrylamide-dT into the thrombin binding aptamer (TBA). Different positions in the TBA sequence were chosen, including internal T sites and endfunctionalisation, as shown in Table 1.…”