Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes

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 <i>N</i>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

Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes

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 <i>N</i>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

Minimal Thioflavin T Modifications Improve Visual Discrimination of Guanine-Quadruplex Topologies and Alter Compound-Induced Topological Structures

We newly synthesized thioflavin T (ThT) analogs for which the methyl group at the <i>N</i>3 position on the benzothiazole ring was replaced with either a ((<i>p</i>-(dimethylamino)­benzoyl)­oxy)­ethyl group (ThT-DB) or a hydroxyethyl group (ThT-HE). In several neutral buffers, ThT-HE bound to a parallel guanine-quadruplex (G4) DNA and selectively emitted strong fluorescence at 74- to 240-fold higher intensities than those in the presence of double-stranded DNA (dsDNA), whereas ThT resulted in only 13- to 25-fold higher intensities. Furthermore, circular dichroism (CD) analyses using ThT, ThT-DB, and ThT-HE showed that these compounds could induce topological changes in G4. In addition, the different chemical structures of the <i>N</i>3 substituents could alter a G4–DNA conformation. These results indicate a great potential for <i>N</i>3-substituted ThT analogs as G4 probes and drug leads to achieve gene expression regulation

Real-Time Monitoring of G‑Quadruplex Formation during Transcription

Cotranscriptional folding of an RNA transcript enables formation of metastable RNA structures. Thermodynamic and kinetic properties of RNA G-quadruplex formation have previously been investigated using purified guanine-rich oligonucleotides. Here, we describe a method for analysis of cotranscriptional dynamics of the G-quadruplex formation based on real-time monitoring of the fluorescence of G-quadruplex ligands. For RNA sequences with the potential to form mutually exclusive hairpin or G-quadruplex structures, the efficiency of G-quadruplex formation during transcription depended on position of the hairpin forming sequence. The real-time monitoring enabled evaluation of environmental effects on RNA dynamics, as we demonstrated facilitation of post-transcriptional G-quadruplex formation under molecular crowding conditions. The strategy demonstrated here provides folding insights into the G-quadruplex during transcription that should be involved in gene regulation