With the ultimate goal of creating life in a test tube, researchers have long been trying to create self-replicating enzymes. As one approach to this goal, Gerald Joyce and his colleagues at The Scripps Research Institute have been working with 'R3C' ligase, an RNA enzyme that self-replicates not nucleotide by nucleotide but rather by base-pairing to and joining two RNA oligonucleotides.

The researchers converted R3C into a cross-replicating system in which a plus-strand enzyme puts together two RNA molecules to create a very similar minus-strand enzyme—and this minus-strand enzyme, in turn, creates the plus-strand enzyme. In vitro evolution enabled selection of efficient replicators for this stepwise chain reaction, which is analogous to M.C. Escher's Drawing Hands. But an illusion this was not: with the addition of just fresh oligonucleotides and magnesium, these enzymes self-amplified exponentially at a constant temperature of 42 °C, with a doubling time of about 1 hour (Lincoln and Joyce, 2009).

“The killer app of the replicator is that it's a signal amplifier for molecular events,” explains Joyce, referring to the subsequent incorporation of an RNA aptamer into the enzyme to make the amplification ligand-dependent (Lam and Joyce, 2009). In the presence of the ligand, the aptamer sequence is ordered, as is the catalytic domain of this 'aptazyme', enabling exponential amplification—just as the hand holding the pencil in Escher's drawing is poised to draw the opposite sleeve. But in the absence of the correct ligand, the entire molecule becomes destabilized and the ligase activity is abolished. Joyce compares the assay to quantitative PCR (QPCR): “We can actually measure the concentration [of the target ligand] based on the exponential growth rate [of the aptazyme].”

With aptamer development now becoming a routine process, this system could be used to detect everything from proteins to metabolites. And improvements to this aptazyme system are on the horizon: within the next year, Joyce projects “a doubling time of 10 minutes. But based on the kinetic properties of the molecule, a doubling time of a minute is, I think, feasible.”