Difference between revisions of "Team:Heidelberg/project/hlpd"

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We successfully applied software based design of a communication module to an HRP-F8 fusion DNAzyme.
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We computationally designed a communication module for an HRP-F8 fusion DNAzyme using our software JAWS. Aptamers for ampicillin and ketamine were joined to the stem of the fused F8 DNAzyme to regulate its cleavage activity. The ketamine-binding aptamer was previously designed by our software MAWS to specifically target the anesthetic, while the ampicillin-specific aptamer was previously published. The fusion DNAzymes were tested against their respective ligand in solution, where 1 µM DNAzyme hemine solution was incubated together with the ligand, hydrogen peroxide, and TMB. Reactions were stopped using 1 mM hydrochloric acid. Catalytic activity of the ampicillin-responsive DNAzyme drastically increased upon exposure to its ligand (Fig. iA), indicated by the solution turning deep blue, while the negative control lacking ampicillin was only faintly coloured. To test this detection system in a more realistic setting, the DNAzyme was incubated in energy drink spiked with ampicillin. Here, a fainter change in color, presumably due to suboptimal buffer conditions was observed, with the ampicillin-spiked beverage still showing significant discoloration compared to the pure drink (Fig. iB). From the two DNAzymes designed for ketamine one switched on in the presence of ketamine, while the other switched off. Both produced similar colors after the reaction was stopped (Fig i+1). Thus, our software suite proved successfull in designing a detection system for a drug within less than a day of computation.
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Aptamers for ampicillin and ketamin were joined to the stem of the fused F8 DNAzyme to regulate it's
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cleavage activity. Out of those aptamers, the one against ketamine was previously designed by our  
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software MAWS to specifically target the anesthetic. The resulting fusion DNAzymes generated by JAWS
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were further tested against their respectice ligand in a liquid phase assay, where 1µM DNAzyme hemine
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solution was incubated together with the ligand, hydrogen peroxide and TMB. Reactions were stopped using
+
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1mM hydrochloric acid. For the case of ampicillin the DNAzyme candidate was shown to switch on after exposure
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to its ligand (Fig. iA), the solution turning deep blue, while the negative control was only faintly coloured.
+
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To test this detection system in a more realistic setting, the DNAzyme was incubated in energy drink
+
−
spiked with ampicillin. Here, a fainter change in color, probably due to suboptimal buffer conditions
+
−
was observed, with the ampicillin-spiked beverage still showing significant change in respect to the pure drink (Fig. iB).
+
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From the two DNAzymes designed for Ketamin one switched on in the presence of ketamine, while the other switched off.
+
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Both produced similar colors after stopping the reaction with hydrochloric acid (Fig i+1).
+
−
Thus, our software suite proved successfull in designing a detection system for a drug within less than a day of
+
−
computation.
+
  
 
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Revision as of 02:41, 19 September 2015

Abstract

Results

We computationally designed a communication module for an HRP-F8 fusion DNAzyme using our software JAWS. Aptamers for ampicillin and ketamine were joined to the stem of the fused F8 DNAzyme to regulate its cleavage activity. The ketamine-binding aptamer was previously designed by our software MAWS to specifically target the anesthetic, while the ampicillin-specific aptamer was previously published. The fusion DNAzymes were tested against their respective ligand in solution, where 1 µM DNAzyme hemine solution was incubated together with the ligand, hydrogen peroxide, and TMB. Reactions were stopped using 1 mM hydrochloric acid. Catalytic activity of the ampicillin-responsive DNAzyme drastically increased upon exposure to its ligand (Fig. iA), indicated by the solution turning deep blue, while the negative control lacking ampicillin was only faintly coloured. To test this detection system in a more realistic setting, the DNAzyme was incubated in energy drink spiked with ampicillin. Here, a fainter change in color, presumably due to suboptimal buffer conditions was observed, with the ampicillin-spiked beverage still showing significant discoloration compared to the pure drink (Fig. iB). From the two DNAzymes designed for ketamine one switched on in the presence of ketamine, while the other switched off. Both produced similar colors after the reaction was stopped (Fig i+1). Thus, our software suite proved successfull in designing a detection system for a drug within less than a day of computation.