Synthetic, Switchable Enzymes

Abstract

The construction of switchable, radiation-controlled, aptameric enzymes - "swenzymes" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low-intensity, nonionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate product-capturing aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis.

Keywords: Aptamer; Catalyst; Catalytic antibody; Enzyme; Light; Radiation; Ribozyme; Sound; Synthetic biology.

Figure 1. The principle of the swenzyme

(a) From a large library of aptamers, an aptamer, S1’, is obtained to bind to substrate 1, S1, and a different aptamer, S2’, is obtained to bind to substrate 2, S2. (b) A linker joins the two aptamers covalently so as to obtain a candidate swenzyme that can bind simultaneously to both substrate 1 and substrate 2. (c) The candidate swenzyme is exposed to different frequencies of radiation (red lightning) so as to allow it to catalyze the reaction.

Figure 2. Principle of the aptameric detector of product

The idea is to detect indirectly the presence of the reaction product, P, because P binds to an aptamer, P’, thereby changing the conformation of this aptamer, and it is this changed product aptamer that is then detected. In the absence of product, P, nucleotide sequences b and f bind one another and d is unbound. In the presence of P, which binds a, b and c, the binding of b and f is disrupted; this allows d to bind f and thereby (1) change the conformation of the linker’ in green that binds to the linker in a nearby candidate swenzyme (this is why linker’goes from upside-down to right-way-up) and (2) change the conformation of e to one that can be recognized by the sequence e’, which is attached to a magnetic bead (large red oval). The complex sketches are represented by the simple symbols at the bottom of the figure.

Figure 3. The process of selection of a swenzyme

A viscous mixture of candidate swenzymes, substrates, product detectors, and magnetic beads are exposed to radiation. The swenzyme-catalyzed reaction leads to a locally high concentration of the products around the swenzyme. These products cause the product detector to change conformation and to bind to both the neighboring swenzyme and a sequence on a magnetic bead. The magnetic bead then allows the ensemble of aptamers in the vicinity of the product aptamer (which contains the swenzyme itself to be precipitated. Symbols as in Figures 1 and 2.