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STX Aptazyme J_3

This part is a noncoding single-stranded DNA Aptazyme, this molecule combines 3 modules, an aptamer for recognition of Saxitoxin (related to the red tide), a HRP-mimicking DNAzyme for report the presence of the toxin, and linkers.

Aptazyme

An aptazyme is a ssDNA molecule that can recognize a ligand and catalyze a reaction. The funcionality of this molecule depends on three modules; an aptamer, for recognition of the ligand, a DNAzyme, for catalize the reaction and linkers, fundamental parts that can bind specific zones of the Aptazyme, and with that, those can act as kidnappers of the catalytic zone of the molecule. The linkers are fundamental, because they are placed in such a way that when the aptamer is not interacting with the ligand, the linkers are kidnapping the DNAzyme, altering the conformation of the DNAzyme (see Figure 1A).

When the aptamer interacts with the ligand, a conformational change takes place in the molecule, this conformational change promotes the movement of the linkers from the original place, and in consecuense the DNAzyme is released, with that the DNAzyme can take the catalytic conformation and can catalyse the reaction (see Figure 1B).

Fig.1 Aptazyme Structures. Each cyrcle corresponds to a nucleotide. (A) Expected structure in an inactive conformation of the Aptazyme, you may notice that the linkers are sequestring the DNAzyme module, impeding its correct catalytic conformation. (B) In the presence of the ligand, the Aptamer module adopts its conformation with the ligand, in consecuense the linkers move releasing the DNAzyme, that now can adopt its catalytic conformation. All this process leaves de Aptazyme on its active conformation.

This part (BBa_K2289002) consists on an Aptazyme that can recognise Saxitoxin (STX) and that can catalyse a chromogenic reaction.

Modules

STX M30f Aptamer (Part:BBa_K2289004): It is an aptamer for saxitoxin, developed by Zheng et. al. 2015, this aptamer was selected by apool of aptamers. It has a dissociation constant (Kd) of 0.133 μM. The aptamer's secondary structure is on Figure 2A.

HRP-Mimicking DNAzyme (Part:BBa_K1614007): It is a DNAzyme developed by Travascio et. al. 1998. This DNAzyme, just as its name says, imitates the horseradish peroxidase catalytic activity, using hydrogen peroxide as a substrate and a reducting agent, it has been demonstrated that works well with a chromogenic reactant, ABTS. This DNAzyme needs Hemin to act as a catalyst, it adopts a G-cuadruplex conformation that allows to form a complex with Hemin, that, at the same time enables the DNAzyme to catalyse the reaction (see Figure 2B).

J_3 linkers: These are linkers generated by the JAWS software developed by Team Heidelberg 2015. Each linker binds to a specific zone of the molecule, allowing the inactive conformation of the Aptazyme. Those were calculated by the software to allow both conformations of the Aptazyme, the inactive one, and the active in the presence of the ligand. According to the software analysis, the DNAzyme with J_3 linkers has an inactive free energy of -12.55 Kcal/mol, and an active free energy of -6.76 Kcal/mol, with a free energy difference of 5.79 Kcal/mol.

Fig.2 STX M30f Aptamer and HRP-mimicking DNAzyme. (A) The structure of the M30f aptamer, developed by Zhen et. al. (2015). (B) Reaction used in our lab studies with Aptazymes, the DNAzyme in the G-cuadruplex conformation forms a complex with Hemin, so it can catalyse the reaction of oxidation of ABTS, hydrogen peroxide mediated, the ABTS*- resulting is a chromogenic molecule that allows the optical detection by eye or by spectrophotometer at λ=414 nm

All in one

Inactive fold: Inactive fold: We expect that the molecule folding in the absence of the toxin is the same as predicted by the [http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi RNAfold WebServer] (see Figure 3A), this prediction comes with a minimum free energy of -20.10 Kcal/mol

In the presence of the toxin: The expected conformation of the Aptazyme is on Figure 3B, we expect that when the toxin is present, the aptamer adopts other conformation so the J_3 linkers can move releasing the DNAzyme just as described above.

Fig.3 STX Dynamics.

Kinetics

Sequence and Features

References

X. Zheng, B. Hu, SX. Gao, D.J. Liu, M.J. Sun, B.H. Jiao, L.H. Wang. (2015). A saxitoxin-binding aptamer whith higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation. Toxicon 101. 41-47.

P. Travascio, Y. Li, D. Sen. (1998). DNA-enghanced peroxidase activity of a DNA-aptamer-hemin complex. Chemistry and Biology, Vol 5 No 9. 505-517.

C. Teller, C Shimnron, I. Willner. (2009). Aptamer-DNAzyme Hairpins for Amplified Biosensing. Anal. Chem., 81. 9114-9119.