Team:Heidelberg/Results/Standardization

iGEM Team Heidelberg 2015

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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.

HRP DNAzyme in the AptaBody

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.

On the Southern Blot

Not only can the HRP be used in the AptaBody but the part can be adapted in other systems as well. We could show that the HRP-mimicking DNAzyme can be blotted in a Southern blot and its activity is recovered on the membrane. Thus the HRP-mimicking DNAzyme can be used as an easy and fast readout on a Southern blot. It can be connected to any RNA or DNA via splinted ligationKershaw2012 (Fig. 4). This system can be used to avoid working with radioactive isotopes and also poses an alternative to using fluorescent probes.

Figure 4. DNA or RNA labeled with HRP-mimicking DNAzyme

RNA and DNA could be label with HRP-mimicking DNAzyme via splinted ligation

Software Validation

Figure 5. Switchable HRP DNAzyme for software validation

HRP-mimicking DNAzyme activity is controlled by an aptamer that stems with part of the active site in absence of the ligand. In presence of the ligand the inhibiting stem is destabled and thus the HRP-mimicking DNAzyme's activity is recovered.

On top of that we needed a high-throughput validation system for the verification of our calculated aptamers. For this we also used the HRP-mimicking DNAzyme. The aptamers that were predicted by MAWS were fused with the HRP DNAzyme using JAWS to generate a bi-stable system (Fig.5). Thus the activity of the HRP DNAzyme became dependent on the presence of the ligand that can bind to the aptamer. This way we validated our aptamer candidates.

HRP-based Detection

We developed this system further to minimize the background activity by combining the HRP DNAzyme with an F8 ribozyme.Wang2014 This way the stem that avoids the formation of the G-quadruplex can be designed stronger, as the F8 DNAzyme is able to cleave of a part of this stem. This results into a weakening of the stem that inhibits HRP-mimicking DNAzyme activity and thus to the formation of the G-quadruplex. The F8 DNAzyme was fused with aptamers so that in the presence of the ligand the F8 DNAzyme becomes active which then activates the HRP-mimicking DNAzyme (Fig. 6).

Further possibilities

Similarly to the HRP DNAzyme other DNAzymes like the RNA-cleaving DNAzymes 7-18 and 10- 23 DNAzyme can be modified to become ligand dependent.Wanga2002 Many more candidates like the ones described by Wang et al. can be applied for multiplexed detection of small molecules using our visualization method for western or southern blotting.

 

Figure 6. Low background switchable HRP-mimicking DNAzyme

The activity of the HRP-mimicking DNAzyme is controlled via a strong stem. A part of the stem is cleaved of by a switchable F8 DNAzyme in presence of a specific ligand.