An RNA Aptamer that Induces Transcription

Abstract

We identified an RNA aptamer that induces TetR-controlled gene expression in Escherichia coli when expressed in the cell. The aptamer was found by a combined approach of in vitro selection for TetR binding and in vivo screening for TetR induction. The smallest active aptamer folds into a stem-loop with an internal loop interrupting the stem. Mutational analysis in vivo and in-line probing in vitro reveal this loop to be the protein binding site. The TetR-inducing activity of the aptamer directly correlates with its stability and the best construct is as efficient as the natural inducer tetracycline. Because of its small size, high induction efficiency, and the stability of the TetR aptamer under in vivo conditions, it is well suited to be an alternative RNA-based inducer of TetR-controlled gene expression.

Chemical synthesis using synthetic biology

“…In one successful example, RNA aptamers evolved to bind the tetracycline repressor protein (TetR) in E. coli induce gene expression from a tet promoter to the same extent as tetracycline [9]. Aptamers joined to a self-cleaving ribozyme form allosterically regulated, self-cleaving structures, or aptazymes [10 ].…”

“…At left, AraC* [3 ] is a mutant version of an arabinose-responsive transcriptional regulator created with directed evolution to avoid 'cross-talk' with the lactose-responsive transcriptional regulator, lac repressor (LacI). On the right, expression from the tet promoter begins when an in vitro-selected RNA aptamer binds specifically to the tetracycline repressor protein, TetR [9]. E 1 and E 3 translation occurs once their mRNAs have been transcribed (B).…”

3.3 Synthetic Biology Principles for Engineering Mammalian Designer Cells

“…Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 7/6/15 11:54 AM Many of these artificial RNA aptamers were selected to specifically bind a wide variety of compounds, including small molecules such as antibiotics (e.g., tetracycline, kanamycin [69,70]), other drugs (e.g., theophylline [71]), proteins (e.g., TetR [72]), MS2 coat protein [73], L7Ae [74]), nucleotides (for example, ATP [75]), amino acids (e.g., L-arginine [76]), whole cells (e.g., African trypanosomes [77]), and Burkitt's lymphoma cells [78]. The theophylline aptamer is a widely used RNA aptamer.…”

A FACS-based screening strategy to assess sequence-specific RNA-binding of Pumilio protein variants in E. coli

“…Depending on their function, prokaryotic mRNAs have a mean half-life of less than 7 min (Selinger et al, 2003). To prolong the availability of target RNA boxAB for TetFC we tried to stabilize boxAB by placing stem loop structures on both the 5′-and 3′-end of the transcript (5F-boxAB-3F, supplementary Figures S1C and S6) (Blind et al, 1999;Hunsicker et al, 2009). Production of target RNA was confirmed in Northern blots (supplementary Figure S6E).…”