Final Report Summary - GENETIC NANOPROBES (GENETICALLY ENCODED FLUORESCENT NANOPROBES FOR DETECTION OF RNA INTERACTIONS)
The Genetically Encoded Nanoprobes project (GEN) set out to develop a genetically encoded technological scaffolding infrastructure based on RNA molecules. We based our hypothesis on the fact that once transcribed from DNA, RNA molecules can fold into secondary and tertiary structures, and as a result can be designed to fold into particular three-dimensional conformations. In addition, RNA binding proteins (RBPs) normally bind such secondary structure features, and thus it is theoretically possible to design such 3-D structure studded with binding sites for RBPs. The RBPs in turn can be fused to a host of ligands, enzymes, or other proteins which can facilitate the self-assembly of novel genetically encoded particles for a host of applications. In developing this technology we had to address three crucial questions:
1. How do we encode multiple binding sites for an RBP in one DNA construct?
2. What is the level of specificity of RBPs to their binding sites?
3. How does the specificity depend on secondary structure?
4. How do multiply bound RBPs affect one another?
We hypothesized that a successful resolution to these questions will be critical for establishing the RNA scaffold design rules that we need in order to fulfill this technology's potential.
During the past 4 years, we developed a screen and showed in a pilot study that multiple RBP binding sites can easily be encoded in a one scaffold design, provided that the RBPs binding capability is sufficiently orthogonal (i.e. no cross talk between RBP binding sites). Second, the sequence encoding the binding site itself is flexible, as long as the secondary structure of the site is conserved. Finally, multiple RBPs can bind next to each other, and in some cases stabilize binding cooperatively, while in others the RBPs do not interact.
The next phase of the project is to develop a repository of functional binding sites for a library of RBPs that are sufficiently orthogonal from one another. After that, we can begin designing a whole host of scaffold related applications that will allow to study nano-chemistry (i.e. reaction carried out in tiny volumes), bar-code real-time tracking of multiple genes in single cells, as well as to develop a whole class of novel optical probes that will self-assemble inside living cells. Thus, the RNA scaffolding technology has the potential to not only usher a new age of green nano-chemistry, but also provide an invaluable tool to the life-science community for future research.
1. How do we encode multiple binding sites for an RBP in one DNA construct?
2. What is the level of specificity of RBPs to their binding sites?
3. How does the specificity depend on secondary structure?
4. How do multiply bound RBPs affect one another?
We hypothesized that a successful resolution to these questions will be critical for establishing the RNA scaffold design rules that we need in order to fulfill this technology's potential.
During the past 4 years, we developed a screen and showed in a pilot study that multiple RBP binding sites can easily be encoded in a one scaffold design, provided that the RBPs binding capability is sufficiently orthogonal (i.e. no cross talk between RBP binding sites). Second, the sequence encoding the binding site itself is flexible, as long as the secondary structure of the site is conserved. Finally, multiple RBPs can bind next to each other, and in some cases stabilize binding cooperatively, while in others the RBPs do not interact.
The next phase of the project is to develop a repository of functional binding sites for a library of RBPs that are sufficiently orthogonal from one another. After that, we can begin designing a whole host of scaffold related applications that will allow to study nano-chemistry (i.e. reaction carried out in tiny volumes), bar-code real-time tracking of multiple genes in single cells, as well as to develop a whole class of novel optical probes that will self-assemble inside living cells. Thus, the RNA scaffolding technology has the potential to not only usher a new age of green nano-chemistry, but also provide an invaluable tool to the life-science community for future research.