(18d) Ligand-Responsive Micrornas: Toward Probing and Programming Cellular States

RNA is a flexible substrate for the construction of information processing devices that evaluate the cellular environment and regulate gene expression accordingly. These devices are principally composed of an aptamer, nucleic acids that bind target molecules with high affinity and specificity, and a gene regulatory element such that regulatory activity is modulated by a binding event between the aptamer and its cognate ligand. The gene regulatory element can act through a myriad of mechanisms that have unique dynamics and regulatory strength, although not every mechanism is functional in a given organism. Therefore, utilizing many of these mechanisms will greatly expand the applicability of RNA-based information processing devices.

In this work, we selected microRNAs (miRNAs) as the gene regulatory element. MiRNAs are endogenous small RNAs that silence gene expression through the RNA interference (RNAi) pathway and are present in most eukaryotes, including fungi, plants, and animals. The regulatory capacity of miRNAs is astounding, as recent studies have suggested that miRNAs regulate over 10% of human genes, including genes implicated in carcinogenesis. Recent studies have also revealed the structural requirements for miRNA processing, providing valuable insights that contribute to the design of ligand-responsive miRNAs.

We engineered ligand-responsive miRNAs by incorporating aptamers into the miRNA basal segment. Initial miRNA processing and subsequent activation of RNAi is sensitive to the basal segment secondary structure such that ligand binding inhibits processing, resulting in upregulation of the target gene. Through the development of ligand-responsive miRNAs, we established a modular composition framework that promotes independent replacement of the aptamer and miRNA sequences to change either the recognized ligand or the target gene. This design was extended by inserting multiple ligand-responsive miRNAs into the same transcript for multi-input control of multiple genes with variable extents of gene silencing. We also demonstrated that the dynamic range can be increased by inserting the ligand-responsive miRNA into the target transcript such that miRNA processing and RNAi-mediated silencing are both inhibited upon ligand binding.

Our work demonstrates that miRNAs can be engineered to serve as information processing devices to dynamically monitor and control cellular processes. These devices can serve as in vivo biosensors that provide a dynamic read-out of the intracellular environment or as autonomous control devices that evaluate the environment and regulate gene expression accordingly.