Design of a Minimized pT181 Attenuator Using Shape-Seq

In an era where targeted gene therapy is slowly becoming a reality, the ability to effectively deliver stable gene circuits remains a difficult challenge. One issue is that nucleic acid delivery becomes increasingly inefficient as the size of the delivered gene circuit gets larger. Thus, there is a need to be able to design smaller gene circuitry. In this work, we describe our efforts to minimize a synthetic RNA regulator, informed with structural insights. The regulator we chose for this study is the pT181 attenuator, which controls transcription by alternatively forming a terminated or anti-terminated structure in response to an antisense RNA. We use our newly updated SHAPE-Seq (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension Sequencing) technique to examine the structural features of the attenuator RNA to find regions that are non-essential. By sequential deletion and mutation, we were able to greatly reduce the size of the pT181 attenuator and show that it still functions in a similar fashion to the original design.