Autocatalytic aptazymes enable ligand-dependent exponential amplification of RNA

Abstract

RNA enzymes have been developed that undergo self-sustained replication at a constant temperature in the absence of proteins. These RNA molecules amplify exponentially through a cross-replicative process, whereby two enzymes catalyze each other's synthesis by joining component oligonucleotides. Other RNA enzymes have been made to operate in a ligand-dependent manner by combining a catalytic domain with a ligand-binding domain (aptamer) to produce an 'aptazyme'. The principle of ligand-dependent RNA catalysis has now been extended to the cross-replicating RNA enzymes so that exponential amplification occurs in the presence, but not the absence, of the cognate ligand. The exponential growth rate of the RNA depends on the concentration of the ligand, allowing one to determine the concentration of ligand in a sample. This process is analogous to quantitative PCR (qPCR) but can be generalized to a wide variety of targets, including proteins and small molecules that are relevant to medical diagnostics and environmental monitoring.

Figure 1

Sequence and secondary structure of autocatalytic aptazymes. The complex shown is that of the enzyme E and its substrates A´ and B´. Curved arrow indicates the site of ligation, resulting in formation of E´. The reciprocal reaction, involving the enzyme E´ and substrates A and B, is not shown. Dashed boxes indicate regions that were replaced by either the theophylline or FMN aptamer to form the corresponding aptazymes. Solid boxes indicate regions of Watson-Crick pairing that were replaced to allow multiplexed exponential amplification (the AAGU sequence in A´ was replaced by AGUA; the UGAA sequence in B´ was replaced by AUGA).

Figure 2

Ligand-dependent exponential amplification of RNA. (a) The theophylline-dependent aptazymes, E_theo (black) and E´_theo (gray), amplified exponentially in the presence of 5 mM theophylline (filled circles), but not in the presence of 5 mM caffeine (open circles). The structures of theophylline and caffeine are shown. (b) Exponential growth rate of E_theo in the presence of various concentrations of theophylline. (c) The FMN-dependent aptazymes, E_FMN (black) and E´_FMN (gray), amplified exponentially in the presence of 1 mM FMN. The structure of FMN is shown. (d) Exponential growth rate of E_FMN in the presence of various concentrations of FMN. Growth rates for reactions that did not proceed beyond 10% fraction reacted were determined by a linear rather than exponential fit.

Figure 3

Multiplexed ligand-dependent exponential amplification of RNA. The theophylline-and FMN-dependent aptazymes were made to contain distinct regions of Watson-Crick pairing (Fig. 1). Exponential amplification of E_theo (circles) and E_FMN (squares) occurred in the presence of both ligands (black) and in the presence of their cognate ligand alone (gray), but not in the presence of the non-cognate ligand alone (open symbols). Reaction mixtures contained 0.1 µM E_theo and E´_theo, 0.02 µM E_FMN and E´_FMN, and 5 µM each of the eight corresponding RNA substrates.

Figure 4

Monitoring the course of exponential amplification by a luciferase assay, driven by the release of inorganic pyrophosphate that accompanies RNA ligation. Amplification was carried out in the presence of 5 mM theophylline, and the summed yields of E_theo and E´_theo were measured both by separating the ligated products in a denaturing polyacrylamide gel (filled circles) and based on the luminescent signal generated by an ATP-regenerative luciferase assay (filled squares). Light units were converted to absolute concentrations of inorganic pyrophosphate based on comparison to known standards (Supplementary Fig. 4 online). There was no light signal above background in the absence of theophylline (open squares); slightly negative values are due to imprecision in determining the conversion factor.