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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 proteins1. 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'2,3. 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.

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Figure 1: Sequence and secondary structure of autocatalytic aptazymes.
Figure 2: Ligand-dependent exponential amplification of RNA.
Figure 3: Multiplexed ligand-dependent exponential amplification of RNA.
Figure 4: Monitoring the course of exponential amplification by a luciferase assay driven by the release of inorganic pyrophosphate that accompanies RNA ligation.

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Acknowledgements

This work was supported by grant R01GM065130 from the National Institutes of Health (NIH) and by The Skaggs Institute for Chemical Biology at The Scripps Research Institute. B.J.L. was supported by NIH Ruth L. Kirschstein National Research Service Award 5F32GM078691. We thank William McAllister, State University of New York, Brooklyn, for kindly providing plasmid pBH161 encoding histidine-tagged T7 RNA polymerase.

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Correspondence to Gerald F Joyce.

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A patent application has been filed describing the method of ligand-dependent exponential amplification of RNA.

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Lam, B., Joyce, G. Autocatalytic aptazymes enable ligand-dependent exponential amplification of RNA. Nat Biotechnol 27, 288–292 (2009). https://doi.org/10.1038/nbt.1528

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