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標題: Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator
作者: Lin, Ya-Hui
Chang, Kung-Yao
關鍵字: Animals;Base Sequence;Cell Line;Humans;Inverted Repeat Sequences;Mammals;Models, Biological;Nucleic Acid Conformation;RNA, Viral;SARS Virus;Synthetic Biology;Theophylline;Frameshifting, Ribosomal;Ligands;Riboswitch
Project: Nucleic acids research, Volume 44, Issue 18, Page(s) 9005-9015.
Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate -1 programmed ribosomal frameshifting (PRF), suggesting -1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the -1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent -1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of -1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent -1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA-ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific -1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application.
DOI: 10.1093/nar/gkw718
Appears in Collections:生物化學研究所

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