Detection Systems Using the Ternary Complex Formation of Nucleic Acids

Abstract

As a method for detecting a target nucleic acid strand such as DNA or RNA, polymerase chain reaction (PCR) using a thermal cycler and loop-mediated isothermal amplification (LAMP), which is an isothermal amplification reaction, is widely known. These methods involve the process of nucleic acid amplification to detect trace targets and are generally suitable for the detection of long-stranded nucleic acids. On the other hand, when the target is a short and/or modified strand, the efficiency of nucleic acid amplification can be significantly reduced, which makes the detection difficult. In addition, when the target is RNA, reverse-transcription by reverse-transcriptase is necessary in advance of amplification. Therefore, the development of methodologies to evade these problems has been promoted by using a three-way junction (3WJ) structure having a three-pronged branch point consisting of three nucleic acid strands. The methods reported so far are generally classified into those with and without nucleic acid amplification, and in any method, 3WJ plays an essential role for target detection in molecular recognition (sensing) of the target nucleic acid strand and/or signal amplification. Eventually, signal output can be quantitatively collected by fluorometry using nucleic acid fluorescence staining, electrochemical measurement by redox labeling, colorimetry with dye substrates, etc. In methods involving nucleic acid amplification, various detection systems have been contrived that include signal amplification reactions triggered by the formation of 3WJ containing the target nucleic acid strand, by combining with isothermal amplification methods such as exponential amplification reaction (EXPAR) and/or rolling circle amplification (RCA). Furthermore, combining 3WJs and aptamers enabled to detect non-nucleic acid targets as with immunostaining (ELISA) using antibodies, which shows the possibility of simultaneous quantitative analysis of biomarkers with different chemical species such as nucleic acids, proteins, and small molecules on a single platform.KeywordsAlkaline phosphataseAptamerBranched DNA (bDNA) assayColor reactionColorimetryCyclic voltammetry (CV)DNAzymeElectrochemiluminescence (ECL)Exponential amplification reaction (EXPAR)Field effect transistor substrate with graphene (g-FET)Fluorescent stainingFluorometryGuanine-quadruplex (G4)Horseradish peroxidase (HRP)Isothermal amplificationLoop-mediated isothermal amplification (LAMP)Nicking enzymePolymerase chain reaction (PCR)Raman spectroscopyRolling circle amplification (RCA)Signal amplification by ternary initiation complexes (SATIC)Signal-mediated amplification of RNA technology (SMART)Strand displacementThree-way junction(TWJ，3WJ)