Nucleic acids are frequently used to amplify signals during the detection of both nucleic acids and non-nucleic acid analytes including proteins and small molecules. In applications such as immuno-PCR (1
), target-dependent rolling circle amplification (3
), and the proximity ligation assay (5
), nucleic acids frequently act as captured or created templates for amplification. It is also possible to rely on ribozymes, rather than protein enzymes, for amplification. For example, RNA-cleaving deoxyribozymes (7
) and RNA-ligating ribozymes (9
) have been used to construct autocatalytic or cross-catalytic circuits for exponential signal amplification in the detection of nucleic acid and/or small molecule analytes. While nucleic acid-alone amplification schemes have the advantage of operating under conditions that might otherwise inhibit proteins, they currently have the disadvantage of requiring a great deal of engineering for adaptation to any given analyte. For example, directed evolution of the Bartel Class I ligase was necessary in order to adapt it to detecting a particular mRNA sequence (11
Recent advances in the field of molecular programming (12–15
) have yielded DNA circuits in which the simple rules governing nucleic acid hybridization can be adapted to signal-amplification. The hybridization chain reaction (HCR) (16
), entropy-driven catalysis (18
), and catalyzed hairpin assembly (19
) rely only on hybridization and strand-exchange reactions in order to achieve amplification.
Some hybridization-based, nucleic-acid-alone circuits have been adapted to analytical applications. For example, the Pierce group has successfully used the hybridization chain reaction for multiplexed imaging of endogenous mRNA in fixed zebrafish embryos (17
). The application of HCR and entropy-driven catalysis in assisting in vitro
nucleic acid detection has also been reported (20–22
). Building on these results, we believe that there now exists an even greater potential to adapt catalyzed hairpin assembly (19
) to support even more robust analytical applications.
A key feature of nucleic acid circuits for molecular amplification is that they do not require perishable protein enzymes. Although some enzymes can be stored for a relatively long period of time in a dry or semi-dry state, all oligonucleotides are amenable to such storage. Moreover, nucleic acid circuits are inherently modular and scalable, requiring only the design of base-pairing between strands. However, within this broad context there are several practical analytical features that should be achieved. First, the uncatalyzed reaction must be sufficiently slow to ensure low background. Second, the turnover of substrates must be fast enough so that a high level of signal amplification can be achieved in a relatively short period of time. Third, it should be possible to adapt the catalyst to detect various analytes. And finally, the product of the catalysis should be easily detectable by common detection modalities, such as fluorescence, colorimetric, or electrochemical detection.
In this work, we designed a simplified DNA circuit based on catalyzed hairpin assembly. Kinetic characterization of this circuit showed that the uncatalyzed background assembly was undetectable (<0.5
). This result is particularly noteworthy since uncatalyzed background reactions have been a major challenge in the design of DNA circuits for analytical applications. In particular, the original hairpin assembly circuit (19
) had an uncatalyzed rate constant as high as ~100
. In addition, our circuit was very efficient and steady-state analysis showed that the turnover rate of the catalyst was >1
, yielding 50- to 100-fold signal amplification within a few hours. Finally, the modularity of the DNA circuit allowed its adaptation to detect various analytes. For example, we engineered a molecular beacon (23
) to act as a signal transducer for virtually any nucleic acid sequence, and showed that similar aptamer beacon (25–27
) transducers allowed the detection of non-nucleic acid analytes. The circuit’s modularity also allowed the output to be readily switched between fluorescence, electrochemical (28
), or colorimetric readouts (30–33
). Overall, these data suggest a paradigm for circuit-based molecular detection in which target recognition, signal processing, amplification, and transduction can be integrated via modular components.