Our method for characterizing the cellular structure and function of RNA regulators is out in Nucleic Acids Research. The paper outlines our method for applying SHAPE-Seq to characterize RNA structures within E. coli cells by directly applying the 1M7 SHAPE reagent to cell culture. Since we are very interested in RNAs that regulate gene expression, we can also simultaneously measure fluorescent reporter protein expression within the same cell culture and thereby directly link the measurement of RNA structure and function!
We are very excited by the technique, because it provides exactly the type of structure/function information that we need to engineer some awesome RNA molecules. In fact, we think it will contribute to a new paradigm for RNA engineering whereby this type of structure measurement is coupled with RNA design to have extremely informative and rapid design-build-structure/function test cycles for RNA engineering (outlined in our recent review and summarized in a neat graphic:)
We showcased the in-cell SHAPE-Seq method by applying it to studying the structure of several RNA regulatory molecules that control translation, including some of the classics of synthetic biology. By focusing on translation we could also also start to ask new questions, such as exactly which nucleotides of the ribosome binding site change in reactivity when these mechanisms are activated. We also demonstrate probing on several endogenously expressed E. coli RNAs including 5S rRNA, RNAse P and the AdoCbl riboswitch of the btuB gene which we hope will motivate the use of our technique for lots of fundamental biology studies.
- Supplementary information (including a detailed step-by-step protocol)