Modularity and Standardization
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 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
Initially we have connected the HRP-mimicking DNAzyme via a linker region to a His-tag aptamer
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
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 ligation
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.
We developed this system further to minimize the background activity by combining the HRP DNAzyme with an F8 ribozyme.
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.