Difference between revisions of "Team:UChile Biotec/Results"

To validate the utility of JAWS for generating aptazymes sequences from the DNAzyme HRP mimicking and an aptamer for any other molecule, the sequence of an aptamer for AMP was introduced into the program, with the purpose of contrasting the catalytic activity of this aptazyme to the catalytic activity of an aptazyme that has already been characterized and tested in the literature and also by our team last year (APTZ_AMP_P) [1].

The sequence which its free energy difference had the highest value among all of the sequences given by the software was chosen (first sequence of Annex 1, notebook), because this difference consists in the difference of energies between the inactive and active state, so it is the sequence that has the least probability to move from an inactive state to an active state arbitrarily, avoiding false positives.

In addition, since the software delivers sequences with two DNAzymes, one on each flank of the sequence and adjacent to the generated linkers (because the software was designed this way, see Heidelberg 2015 project for more information), the sequences chosen to synthesize and subsequently carry out the enzymatic tests were chosen as delivered by the program (raw) and truncated, with the DNAzyme of the 3 'flank excised based on the configuration of the aptazyme APTZ_AMP_P.

The same procedure was done for DNAzyme HRP mimicking and aptamers specific for saxitoxin (APT_STX) and oxadaic acid (APT_OX) described in the literature [2][3]. All the sequences extracted from the literature and the ones obtained with JAWS are described in Annex 1 (notebook).

An experiment was carried out to evaluate if the aptazymes created by JAWS presented a dose-dependent activity for the ligand. Reactions were performed to evaluate the catalytic activity of 3 aptazymes of AMP: the already characterized aptazyme (APTZ_AMP_P) on which there is already evidence of a dose dependence of the ligand, an aptazyme obtained by the JAWS program that has 2 DNAzymes, one in the 5 'end and another at the 3' end of the sequence (APTZ_AMP_J), and the same aptazyme obtained by JAWS but with one of the DNAzymes cut out from the sequence, located at 3 '(APTZ_AMP_JC). It was decided to cut the DNAzyme from this end to emulate the configuration of the aptazyme characterized in the literature.

The assays were performed in Flat-bottomed 96-well plates at different concentrations of triplicate AMP for each sequence and included a positive control; a triplicate of DNAzyme. The experimental scheme is presented in figure 1. Table I (notebook) shows the final concentration of each reagent in 100 μL and the volume in μL.

Here, the sequences worked in addition to the APTZ_AMP_P are presented:

GGGTAGGGCGGGTTGGGgataaccttcCCTGGGGGAGTATTGCGGAGGAAGGTTatccccagcgGGGTAGGGCGGGTTGGG

GGGTAGGGCGGGTTGGGgataaccttcCCTGGGGGAGTATTGCGGAGGAAGGTTatccccagcg

See protocol in the notebook

Results of each tests are presented in figures 2, 3 and 4 for aptazymes “APTZ_AMP_P”, “APTZ_AMP_J” and “APTZ_AMP_JC” respectively, where the absorbance values ​​have been plotted as function of time with duplicate for the first aptazyme and triplicated for the last two.

From these graphs we could conclude that the aptazyme “APTZ_AMP_JC” presents a dose dependence as does the “APTZ_AMP_P” aptazyme, while “APTZ_AMP_J” aptazyme did not have dose dependence. However the “APTZ_AMP_JC” aptazyme presented a greater range of error and / or variability than “APTZ_AMP_P” aptazyme.