(116a) A Systems Strategy for Engineering Families of Orthogonal RNA Transcription Regulators for Engineering Gene Networks

A central goal of synthetic biology is to develop a toolkit of building blocks that can be used to engineer whole biomolecular and cellular systems from the bottom up.  A key component of this toolkit are biological parts that regulate gene expression in ways that can be used to construct networks that integrate and propagate cellular information according to design.  Recently, non-coding RNA-based gene regulatory mechanisms have emerged as powerful and versatile substrates for regulatory parts development [1-3].  One example of an RNA-based regulator is the transcription attenuator engineered from the S. aureus plasmid pT181 [4].  This antisense RNA-mediated mechanism has already been shown to be able to evaluate genetic NOR-like logics in front of a single target, and is the only RNA-based mechanism that can directly propagate RNA signals in simple transcriptional networks.  However, the construction of more sophisticated RNA-based circuitry requires larger numbers of orthogonally acting attenuators for independent control of multiple targets within the network design.  Interestingly, the recent discovery by systems biologists that bacterial genetic networks commonly utilize multiple non-coding RNA regulators in critical functional positions [5] suggests that nature may have already solved the RNA regulator orthogonality problem. We have therefore devised a strategy to expand our library of attenuators by creating chimeric fusions between the pT181 attenuator transcriptional expression platform, and RNA binding domains from other families of natural non-coding RNAs.

The pT181 attenuator is an RNA sequence in the 5’ untranslated region of a transcript that regulates transcription elongation through structural changes mediated by antisense-RNA binding.  This binding is initiated between a loop structure of the attenuator, and the complementary loop structure of the antisense RNA.  Therefore, we hypothesized that new orthogonal chimeric attenuators could be engineered by replacing the RNA-loop sensing region of the pT181 attenuator with RNA-loop sensing regions from a number of naturally occurring antisense RNA-mediated translation regulatory mechanisms.  A systematic investigation of fusion position with three different translation systems that utilize loop-loop interactions led to the addition of two orthogonal chimeric attenuators to the existing library.  In addition, we created an orthogonal chimeric fusion from a translation mechanism that utilizes a loop-linear RNA-RNA interaction mechanism. Structural analysis of both functioning and non-functioning chimeric attenuators with a high-throughput chemical probing technique called SHAPE-Seq revealed insights into the structural requirements of the chimeric junction.  This work is leading to the development of RNA structure/function design rules that will allow the creation of a bioinformatic systems approach to search through natural RNA networks to computationally design larger numbers of orthogonal RNA transcription regulators. This will expand our ability to construct larger synthetic RNA networks, as well as contribute to a systems-level understanding of RNA structure/function modularity that will lead to a deeper understanding of RNA’s role in biology. 

[1] Buskirk, A. R., Kehayova, P. D., Landrigan, A., Liu, D. R., In vivo evolution of an RNA-based transcriptional activator. Chem Biol 2003, 10, 533-540.

[2] Bayer, T. S., Smolke, C. D., Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol 2005, 23, 337-343.

[3] Isaacs, F. J., Dwyer, D. J., Ding, C., Pervouchine, D. D., et al., Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol 2004, 22, 841-847.

[4] Lucks, J. B., Qi, L., Mutalik, V. K., Wang, D., Arkin, A. P., Versatile RNA-sensing transcriptional regulators for engineering genetic networks. PNAS 2011, 108, 8617-8622.

[5] Gottesman, S., Storz, G., Bacterial Small RNA Regulators: Versatile Roles and Rapidly Evolving Variations. Cold Spring Harb Perspect Biol 2011, 3, 1-16.