Aptamer‐Array‐Guided Protein Assembly Enhances Synthetic mRNA Switch Performance

Abstract

Synthetic messenger RNA (mRNA) switches are powerful synthetic biological tools that can sense cellular molecules to manipulate cell fate. However, their performances are limited by high output signal noise due to leaky output protein expression. Here, we designed a readout control module that disables protein leakage from generating signal. Aptamer array on the switch guides the inactive output protein to selfassemble into functional assemblies that generate output signal. Leaky protein expression fails to saturate the array, thus produces marginal signal. In this study, we demonstrated that switches with this module exhibit substantially lower signal noise and, consequently, higher input sensitivity and wider output range. Such switches are applicable for different types of input molecules and output proteins. The work here demonstrates a new type of spatially guided protein selfassembly, affording novel synthetic mRNA switches that promise accurate cell manipulation for biomedical applications.

Dual Input-Controlled Synthetic mRNA Circuit for Bidirectional Protein Expression Regulation

“…The Luc ORF sequence was obtained from plasmid pTK305 (Addgene#66812). The mRNAs were synthesized following the MegaScript T7 Kit (Invitrogen) protocol . The RNA products were purified using the RNeasy MinElute Cleanup kit (QIAGEN) following the manufacturer’s protocol and then treated with Antarctic Phosphatase (New England Biolabs) at 37 °C for 30 min.…”

“…Twenty-four hours after transfection, the cells were trypsinized and suspended in culture media. Flow cytometry recording and analysis were performed according to the previous work (Attune Nxt flow cytometer or BD FACS Aria III) . The arbitrary units of fluorescent intensity were calibrated using standardized fluorescent beads (Attune Performance Tracking Beads from Invitrogen or CS&T Research Beads from BD) according to the manufacturer’s instructions.…”

“…The mRNAs were synthesized following the MegaScript T7 Kit (Invitrogen) protocol. 36 The RNA products were purified using the RNeasy MinElute Cleanup kit (QIAGEN) following the manufacturer’s protocol and then treated with Antarctic Phosphatase (New England Biolabs) at 37 °C for 30 min. The amounts and purity of the product mRNAs were determined by a NanoVue Plus Spectrophotometer (GE Healthcare) and urea polyacrylamide gel electrophoresis.…”

VPg-based bidirectional synthetic mRNA circuits enable orthogonal protein regulation for high-resolution cell separation

“…[1][2][3][4][5] They can sense various cellular biomarker molecules (e.g., microRNA, protein, ligands) and report the information by altering the expression levels of encoded fluorescent reporter proteins. [3][4][5][6] Because they promise a temporal cellular effect and no damage to genome integrity, these switches have been widely used to identify and separate cells, 7,8 especially stem cell-derived cells, such as neurons and cardiomyocytes. 1,2,9 However, current synthetic mRNA circuits mainly turn on or turn off output protein expression upon input-sensing, resolving cell separation in one direction.…”

“…miR-21 was chosen as the target microRNA, as it is an established cell-type marker microRNA for HepG2, and HeLa cells 2,8,16 and with HEK293 being known to have low miR-21 expression. 7,16 To optimize the miR-21 sensitivity, we constructed miR-21-sensing MCP-VPg mRNAs carrying native nucleotides or modified nucleotides that were used to boost the synthetic mRNA performance. 8,15 To our surprise, the modified nucleotides failed to increase the effect of miR-21 mimic on the EGFP expression from both reporter mRNAs (Fig.…”