Part:BBa_K2289000

STX Aptazyme J_1

Notice: Functional DNA

This part is a sequence of a functional ssDNA. It is only active as single-stranded DNA. It can not be cloned into a plasmid. For use order it as a DNA oligo.

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

Aptazyme

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 the recognition of a ligand, a DNAzyme, htat catalize a reaction and linkers, fundamental parts that can be complementary to specific zones of the Aptazyme, sequestring the catalytic zone of the molecule. The linkers are eseential, because they are placed in such a way that when the aptamer is not interacting with the ligand, the linkers are capturing the DNAzyme and 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 consequently the DNAzyme is released. Then, the DNAzyme can structure the catalytic conformation and catalyze the reaction (see Figure 1B).

Fig.1 Aptazyme Conformational Changes. Each circle corresponds to a nucleotide. (A) Expected structure in an inactive conformation of the Aptazyme, you may notice that the linkers are sequestering the DNAzyme module, impeding its correct catalytic conformation. (B) In the presence of the ligand, the Aptamer module adopts its conformation with the ligand; consequently 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_K2289000) consists on an Aptazyme that can recognise Saxitoxin (STX) and that can catalyse a chromogenic reaction with HRP-like enzymatic activity.

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 reducing agent, it has been demonstrated that works well with a chromogenic reactant, ABTS. This DNAzyme needs Hemin to act as a catalyst, and when adopts a G-cuadruplex conformation allows the formation of a complex with Hemin, that, at the same time enables the DNAzyme to catalyse the reaction (see Figure 2B).

• J_1 linkers:

These are linkers generated by the JAWS software developed by Team Heidelberg iGEM 2015. Each linker binds to a specific zone of the molecule, allowing the inactive conformation of the Aptazyme. Those parameters 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_1 linkers has an inactive free energy of -13.09 Kcal/mol, and an active free energy of -6.11 Kcal/mol, with a free energy difference of 6.98 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 catalyze 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 -19.40 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_1 linkers can move releasing the DNAzyme just as described above.

Fig.3 STX Aptazyme J_1 conformational changes. Each circle corresponds to a nucleotide. (A) Predicted structure of the Aptazyme on its inactive conformation. (B) We expect that in the presence of the toxin, the Aptazyme changes its inactive conformation to its active conformation, the structure of the aptamer interacting with the toxin that you can see is only representative because it is unknown.

Kinetics

• Conditions: In order to evaluate the kinetics of our parts, we determined the amounts of each reactive present in the reaction, see table I, and we determined Fig 4, with those

concentrations.

Table I List of reactants and their concentrations in a total volume of reaction of 100 µL. We also used HEPES buffer 10 mM with 10 mM NaCl pH 7.1.

For more information about the methods used for these experiments, please visit [http://2017.igem.org/Team:UChile_Biotec/Experiments Protocols] in the Experiments section of our Wiki.

• Results:

Fig. 4 Results of the experiments on the STX Aptazyme J_1.

The kinetic results of the Aptazyme are in Figure 4, a qualitative interpretation can be made, as you can see in the figure, there is an increase of absorbance as time progresses, so we can say that the DNAzyme module is working as described above. Now, if you can notice, changes in the concentration of the toxin do change the absorbance curves, but the curves indicate that there is a slight difference at different concentrations, not as we expected. Moreover, in absence of the toxin the Aptazyme has a significant activity. Surprisingly this Aptazyme has more activity than the DNAzyme alone (data not shown).

These results indicate that there is no major difference in the activity of the Aptazyme between the different concentrations of the toxin (including the absence of the toxin); this might happen because of the predicted inactive conformations of the molecule where wrong and the inactive structure could not sequestrate efficiently the DNAzyme module generating a considerable noise. But we worked on very small concentrations and the differences between them are small, so the differences between the curves are slight too.

For more information about results, please go to the [http://2017.igem.org/Team:UChile_Biotec/Results Results] section in our [http://2017.igem.org/Team:UChile_Biotec Wiki].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.