title: Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes.
abstract: We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string. The G4s can be specifically fluorescence-stained with N(3)-hydroxyethyl thioflavin T (ThT-HE), which emits weakly with single- and double-stranded RNA/DNA but strongly with parallel G4s. An improved dual SATIC system, which involves the formation of two different ternary initiation complexes in the RCA process, exhibited a wide quantitative detection range of 1-5000 pM. Furthermore, this enabled visual observation-based RNA detection, which is more rapid and convenient than conventional isothermal methods, such as reverse transcription-loop-mediated isothermal amplification, signal mediated amplification of RNA technology, and RNA-primed rolling circle amplification. Thus, SATIC methodology may serve as an on-site and real-time measurement technique for transcriptomic biomarkers for various diseases.
authors.affiliation: Graduate School of Science and Technology, Gunma University , 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Hiroto
authors.initials: H
authors.lastname: Fujita
authors.affiliation: Graduate School of Science and Technology, Gunma University , 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Kataoka
authors.affiliation: Graduate School of Science and Technology, Gunma University , 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Tobita
authors.affiliation: Graduate School of Science and Technology, Gunma University , 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Kuwahara
authors.forename: Naoki
authors.initials: N
authors.lastname: Sugimoto
mesh_terms: D015415:Biomarkers / Q000032:analysis*; D004270:DNA, Circular / Q000235:genetics*; D054856:G-Quadruplexes; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012313:RNA / Q000235:genetics*
chemical_list: D015415:Biomarkers; D004270:DNA, Circular; D012313:RNA