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.

RNA Biomarkers: Diagnostic and Prognostic Potentials and Recent Developments of Electrochemical Biosensors

“…Copyright 2005, Oxford University Press. II) Reproduced with permission . Copyright 2016, American Chemical Society.…”

“…On the contrary, the RPRCA method uniquely uses RNA primers instead of DNA primers to avoid the use of reverse transcriptase for detecting small RNAs in a single tube . In RPRCA, usually the target RNA hybridizes to its complementary sequence on a circular DNA template, and is then cleaved by RNase H (30 °C) followed by elongation (via ϕ29 DNA polymerase at 30 °C), amplification, and detection steps . In 2016, Fujita et al developed another RCA‐based method referred to as signal amplification by ternary initiation complexes (SATIC) (Figure II).…”

DNA nanotechnology for nucleic acid analysis: multifunctional molecular DNA machine for RNA detection

“…A number of isothermal techniques including loop-mediated (LAMP) 15 a and rolling circle (RCA) 15 b – d amplifications have been used for the detection of DNA and RNA. The deoxyribozyme-based technology amplifies the signal, not the target, and, therefore, senses directly RNA rather than DNA amplicons.…”

Interlinked DNA nano-circles for measuring topoisomerase II activity at the level of single decatenation events

“…Some of these have been successfully integrated with more complex nano-structures (11–14). Many of the sensors or sensor systems are built as relatively simple structures, exemplified by single- or double-stranded DNA structures containing F örster r esonance e nergy t ransfer (FRET) pairs or DNA sequences that fold (or unfold) into DNAzyme or triplex structures upon external stimuli (2,9,15,16). More recently, a robust and fully reversible logic circuit was constructed by a more complex interlocked DNA nanostructure forming a double-stranded DNA pseudo-catenane (5).…”