(599y) in vivo SHAPE-Seq: a method to determine the in-cell structure of RNA

With the growth of genetic circuits and genetic regulation of increasing complexity, there is an even
greater need for more precise and accurate regulatory systems. Of growing interest are synthetic RNA
regulators and the need to engineer more precise, orthogonal systems. One of the greatest bottlenecks to
this engineering is a lack of understanding of how these RNA regulators fold inside the cell, which has
hampered efforts of their engineering. Herein, we present in vivo SHAPE-Seq, a coupling between nextgeneration
sequencing and SHAPE probing of RNA regulatory molecules in E. coli. We began the
development of in-cell SHAPE-Seq by first creating a standardized platform for expressing regulatory
RNAs that is capable of simultaneously measuring regulator function via a fluorescence assay and RNA
structure with SHAPE-Seq. In-cell SHAPE-Seq performs chemical probing of RNAs directly in the cell
utilizing 1M7, and then uses NGS to concurrently probe multiple RNAs while reducing bias to off-target
cDNAs generated. In this poster I present our method of constructing our platform for in vivo SHAPESeq
as well as our method for modifying, extracting, and preparing RNA and cDNA libraries for NGS.
By providing quantitative RNA structural data for in vivo RNA folds, we anticipate in-cell SHAPE-Seq to
motivate an entirely new approach to designing synthetic regulators that uses nucleotide-resolution
structure/function relationships to uncover true RNA design principles. We expect that this type of
characterization feedback at both the structure and function level will greatly speed the design of new
regulators and become a staple of synthetic biology.