title: A novel strategy to analyze L-tryptophan through allosteric Trp repressor based on rolling circle amplification.
abstract: Rolling circle amplification (RCA) has been considered as a powerful tool for nucleic acids detection. Here, a novel repressor-RCA-based method for L-tryptophan (L-Trp) detection was developed. This method utilizes the specific interaction between the RCA circular template and the Trp repressor protein (TrpR) involved in trp operon of Escherichia coli (E. coli). In the absence of L-Trp, the TrpR protein could not bind to the RCA template, and the RCA process can be continued. When L-Trp is present, the activated TrpR will bind to the operon sequence on the RCA template and inhibit the RCA reaction. Thus, the concentration of L-Trp is correlated directly with the fluorescent RCA signals. We succeeded in detecting L-Trp in a single step in simple homogeneous reaction system. The detection limit was estimated to be 0.77 μM (S/N=3) with good linearity. The method can unambiguously distinguish L-Trp from other 19 standard amino acids and L-Trp analogs. This strategy is also promising for detecting many small molecules such as other amino acids and carbohydrates.
authors.affiliation: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning 110001, China.
authors.forename: Guojie
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning 110001, China.
authors.forename: Tianyu
authors.initials: T
authors.lastname: Hu
authors.affiliation: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning 110001, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Animal Science and Veterinary Medicine College, Shenyang Agricultural University, Shenyang, Liaoning 110866, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Wei
authors.affiliation: Department of Clinical Diagnosis, the First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Shang
authors.affiliation: Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning 110001, China. Electronic address: yfguan@mail.cmu.edu.cn.
authors.forename: Yifu
authors.initials: Y
authors.lastname: Guan
mesh_terms: D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D057230:Limit of Detection; D008958:Models, Molecular; D009876:Operon; D012097:Repressor Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins
keywords: Allosteric effect; Repressor; Rolling circle amplification; Tryptophan detection