Difference between revisions of "Part:BBa K2289003"

 
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<partinfo>BBa_K2289003 short</partinfo>
 
<partinfo>BBa_K2289003 short</partinfo>
  
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This part is a noncoding single-stranded DNA Aptazyme, this molecule combines 3 modules, an HRP-mimicking DNAzyme, an aptamer for recognition of Saxitoxin (related to the red tide) and linkers.
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{{Template:ssDNA}}<br/>
 +
 
 +
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 promote 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).
 +
 
 +
[[File:UchileBiotec2017Figure1.png|thumb|500 px|center|'''Fig.1 Aptazyme.''']]
 +
 
 +
This part consists on an Aptazyme that can recognise Saxitoxin (STX) and that can catalyse a chromogenic reaction.
 +
 
 +
==Modules==
 +
 
 +
<b> STX M30f Aptamer ([https://parts.igem.org/Part:BBa_K2289004 Part:BBa_K2289004]): </b>
 +
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 uM. The aptamer's secondary structure is on Figure 2A.
 +
 
 +
<b> HRP-Mimicking DNAzyme ([https://parts.igem.org/Part:BBa_K1614007 Part:BBa_K1614007]): </b>
 +
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).
 +
 
 +
<b> J_4 linkers: </b>
 +
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.
 +
 
 +
[[File:UchileBiotec2017Figure2.png|thumb|500 px|center|'''Fig.2 Aptamer and DNAzyme.''']]
 +
 
 +
==All in one==
 +
 
 +
<b> Inactive fold: </b> <b> Inactive fold: </b> 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]
 +
 +
 
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<b> In the presence of the toxin: </b>
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<b> Dynamics and kinetics: </b>
  
 
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<span class='h3bb'>Sequence and Features</span>
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==<b>Sequence and Features</b>==
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<partinfo>BBa_K2289003 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2289000 SequenceAndFeatures</partinfo>
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==References==
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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.
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2289003 parameters</partinfo>
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<partinfo>BBa_K2289000 parameters</partinfo>
 
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Revision as of 08:08, 29 October 2017


STX Aptazyme J_4

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 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 promote 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.

This part 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 uM. 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_4 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.

Fig.2 Aptamer and DNAzyme.

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]


In the presence of the toxin:

Dynamics and kinetics:

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.