In Vitro Reversible Translation Control Using γPNA Probes

Abstract

On-demand regulation of gene expression in living cells is a central goal of chemical biology and antisense therapeutic development. While significant advances have allowed regulatory modulation through inserted genetic elements, on-demand control of the expression/translation state of a given native gene by complementary sequence interactions remains a technical challenge. Toward this objective, we demonstrate the reversible suppression of a luciferase gene in cell-free translation using Watson-Crick base pairing between the mRNA and a complementary γ-modified peptide nucleic acid (γPNA) sequence with a noncomplementary toehold. Exploiting the favorable thermodynamics of γPNA-γPNA interactions, the antisense sequence can be removed by hybridization of a second, fully complementary γPNA, through a strand displacement reaction, allowing translation to proceed. Complementary RNA is also shown to displace the bound antisense γPNA, opening up possibilities of in vivo regulation by native gene expression.

A modular clamp-like mechanism to regulate the activity of nucleic-acid target-responsive nanoswitches with external activators

“…For example, a similar strategy could be applied in the field of synthetic biology to design genetic circuits that, through different modulators, can finely control biological pathways and cellular functions including transcription and gene expression. [30][31][32][33][34][35][36][37][58][59] …”

Nucleic Acids and Their Analogues for Biomedical Applications

“…In addition, GPNA was less cytotoxic compared with PNA-polyarginine conjugates. In a recent study, Bruchez et al demonstrated the reversible suppression of a luciferase gene using a γ-modified PNA (γPNA) sequence and a non-complementary toehold [ 91 ]. The antisense γPNA strand could be removed by a second, fully complementary γPNA strand via a strand displacement reaction, leading to the continuation of translation.…”

Peptide nucleic acids and their role in gene regulation and editing

“…While significant advances have been made toward the regulation of gene expression by manipulation of genetic elements, on demand control of transcription/translation of a desired gene with sequence specificity remains a challenge. [ 40 ] As a step toward this goal, Canady et al . demonstrated the reversible suppression of a luciferase gene in cell‐free translation, by targeting a mRNA transcript using a complementary γPNA sequence with a non‐complementary toehold.…”

“…Furthermore, the authors showed that instead of γPNA, complementary RNA can also displace the bound antisense γPNA, presenting possibilities of regulation involving the native cellular gene expression pathways. [ 40 ] If developed to be applicable within in vitro and in vivo settings, such a tool would be very useful in the field of modulating gene expression.…”