Collections/Functional Nucleic Acids/Aptazymes

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Aptazymes: modularly engineered nucleic acid enzymes

Aptazymes are ligand-dependent self-cleaving ribozymes (J. Tang & Breaker, 1997). They consist of three basic parts: an aptamer as a ligand binding domain, a self-cleaving ribozyme, and a communication module between them. The communication module typically dislocates some nucleotides from the catalytic site of the self-cleaving ribozyme. Only upon ligand binding, a conformational change to the communication module enables ribozyme self-cleavage. The latter property, coupled to their high modularity, render aptazymes as an important element of the synthetic biology toolbox.

Aptazymes display some advantages over riboswitches. Firstly, they are not restricted to the 5’ end of mRNA molecules, which is the case for riboswitches. Second,they can be used to regulate a variety of different RNA molecules given their modular and programmable nature. For example, aptazymes can often be engineered to encode at the 3’ end of mRNAs, where, upon ligand binding, they induce degradation of the targeted transcript (Nomura et al., 2012; Yen et al., 2004). Moreover, aptazymes have been recently incorporated in guide RNAs for ligand-responsive gene editing with CRISPR/Cas9 (W. Tang et al., 2017), and their versatile nature allows them to be easily transformed into logic gates (Nomura & Yokobayashi, 2015). Finally, since they are underpinned by aptamers, they can be easily designed to bind virtually any target molecule.

Aptazymes are among the class of FNAs that have not been broadly incorporated into iGEM projects. However, several teams have attempted to couple them with their systems. Team Strasbourg 2019, for instance, designed an aptazyme that consisted of two protein aptamer domains and one guanine sensing motif. The protein-recognising domains could bind repressor proteins and recruit them to an operator. In their system, when both of the repressor proteins are bound to the operator, the expression of LacZ gene located downstream is inhibited. However, in the presence of guanine, it binds to the G-sensing aptamer and mediates cleavage of the aptazyme, thus allowing one of the repressor proteins to dissociate from the complex. In turn, this enables the expression of LacZ and therefore turns the system ON, showcasing their design aimed at engineering a platform for food allergen detection.

References

Tang J, Breaker RR. Rational design of allosteric ribozymes. Chem Biol. 1997 Jun;4(6):453-9. https://doi.org/10.1016/s1074-5521(97)90197-6.

Nomura Y, Kumar D, Yokobayashi Y. Synthetic mammalian riboswitches based on guanine aptazyme. Chem Commun (Camb). 2012 Jul 21;48(57):7215-7. https://doi.org/10.1039/c2cc33140c.

Yen L, Svendsen J, Lee JS, Gray JT, Magnier M, Baba T, D’Amato RJ, Mulligan RC. 2004. Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 431:471–476. doi: 10.1038/nature02844. Nomura Y, Yokobayashi Y. Aptazyme-based riboswitches and logic gates in mammalian cells. Methods Mol Biol. 2015;1316:141-8. https://doi.org/10.1007/978-1-4939-2730-2_12.