Gene therapy, for all its recent progress, seems a bit stuck on the idea of introducing, well, genes, meaning DNA sequences. But what about that other nucleic acid, RNA? RNA potentially has several advantages over DNA, including a much lower risk of genome integration and lower immunogenicity. In fact, we recently saw a transcription-free CRISPR-cas9 system. Chemically modified RNA can also be very stable and, with appropriate RNA polymerases, can self-replicate, allowing long-term gene expression.
RNA-RNA-Binding Protein Repression Circuits
However, complex DNA-less genetic circuits had not been shown in mammalian cells until a recent collaboration between scientists at MIT and Kyoto University. For this demonstration, lead author Liliana Wroblewska and colleagues first characterized a set of DNA-less, post-transcriptional circuit parts, consisting of RNA-binding proteins (RBPs) and their RNA binding motifs. When the RBP binds its target RNA, it represses translation of the output (here, GFP).
DNA-Free HeLa Cancer Detection
Of course, this circuit still relies on normal translation, so it can respond to any other form of post-transcriptional regulation, such as microRNAs. Conveniently, the team had previously developed just such a post-transcriptional microRNA-sensing circuit that can accurately detect when it is inside a HeLa cancer cell.
After finding good RBP repressors, the team used them to modify their HeLa cell classifier to work sans DNA. First, they designed an output RNA to be repressed by three microRNAs that have low abundance in HeLa cells. For a microRNA that is highly expressed in HeLa cells, they included another RNA encoding an RBP that would also repress the output, but which was itself repressed by the HeLa-high microRNA (double inversion, yeah, you may need to sketch it out). The circuit did a nice job of distinguishing HeLa cells from benign HEK cells, and it could even kill the cancerous HeLas.
Moving beyond this classifier, the team made a 3-stage repression cascade and even a toggle switch that can be switched between two states – A high, B low and A low, B high – by a pair of siRNAs.
So don’t let DNA keep you down! Get some more oxygen; join the 2’-hydroxy revolution over at Nature Biotechnology, 2015.