Preparation ofE. coliRNA polymerase transcription elongation complexes for systematic RNA assays

RNA folds into secondary and tertiary structures that can mediate diverse cellular functions. Understanding how RNA sequence directs the formation of biologically active structures requires approaches that can comprehensively assess how changes in an RNA sequence affect its structure and function. Towards this goal, I have developed a general method for purifying E. coli RNA polymerase (RNAP) transcription elongation complexes (TECs) for use in systematic RNA assays. My approach depends on two constituent technologies: First, I have designed an E. coli σ 70 promoter that can be efficiently barcoded using a one-pot series of enzymatic reactions. Second, I have developed a strategy for purifying promoter-initiated E. coli RNAP TECs by selective photo-elution from streptavidin-coated magnetic beads. Together, these methods establish a platform for the development of TEC Display assays in which the functional properties of RNA sequence variants can be recorded by fractionating and quantitatively barcoding a TEC library.