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Revision as of 16:22, 18 September 2015

Modularity and Standardization

Figure 1. Modular design of aptazymes

An aptamer is connected via a communication module to catalytic sequence. Upon binding of the ligand the communication module transduces a change to the catalytic site and results in activity.

As functional DNA in iGEM has been rarely mentioned we want to introduce it to iGEM. We want to provide standard parts that everyone can use to make the work with functional DNA and also RNA accessible for the whole community. To simplify RNA work we designed a new BBF RFC 110 to standardize the processes necessary for this.

This new BBF RFC 110 is very important for the work with RNA as the cloning strategy does not rely on any restriction site. We designed it this way because if you work with a functional RNA the sequence is extremely essential. In most cases the sequence is laboriously optimized with several cycles of SELEX. If the functional RNA contains a cut sites usually one cannot simply mutate it as it is possible for proteins. Any mutation alters the effector sequence directly and thus can have severe effects on the functionality.

Furthermore we have standardized the work with functional DNA. Has functional DNA usually is ssDNA it can NOT be cloned into a plasmid for storage. But every single strand functional DNA can simply be ordered as oligos.

In our project we mainly combined aptamer with a catalytic nucleic acid and thus create an aptazyme as symbolized in Fig. 1. However some constructs are more complicated than this (see HRP based detection). The parts we use can of cause be used in different constructs as well and is not limited to the setup we propose.

HRP-mimicking DNAzyme

HRP-mimicking DNAzyme folds into a G-quadruplex and binds hemin into it. Upon binding of the hemin to the G-quadruplex the DNAzyme catalyzes the reduction of H2O2 to H2O and a reactive oxygen species and thus result to the activation of a classical HRP substrate like luminol.

 

HRP DNAzyme in the AptaBody

Figure 3. AptaBody

An aptamer connected via a linker to a HRP-mimicking DNAzyme that detects protein an a Western blot.

Initially we have connected the HRP-mimicking DNAzyme via a linker region to a His-tag aptamerBartnicki2014 (Fig.3). This way we showed that these two parts together with a linker that connects both can be applied to detect different His-tagged proteins from cell lysate on a Western blot. The aptamer part of this construct can easily be exchange by any other aptamer as we could show for p53. We generated an aptamer for p53 and were able to detect p53 with its AptaBody. We have calculated several other aptamers for proteins that need to be tested with our software MAWS. Aptamers generated by our software MAWS can be fused to the versatile HRP DNAzyme to produce a library of AptaBodies.

The transformation of a terminal label into an internal label, one can be achieved by splinted ligation using a DNA template that is complementary to the two RNA templates that are to be connected to each other Kershaw2012.

Figure 2. HRP-micking DNAzyme

HRP-mimicking DNAzyme folds into a G-quadruplex and binds hemin into it (Fig. 2). Upon binding of the hemin to the G-quadruplex the DNAzyme catalyzes the reduction of H2O2 to H2O and a reactive oxygen species and thus result to the activation of a classical HRP substrate like luminol.