Hybridization Cascade Plus Strand-Displacement Isothermal Amplification of RNA Target with Secondary Structure Motifs and Its Application for Detecting Dengue and Zika Viruses

Abstract

Biological RNA generally comprises secondary structure motifs which cause a problem for target RNA detection by isothermal amplification methods. The complexity of the secondary structures makes RNA targets inaccessible for probe hybridization, resulting in decreased sensitivity and selectivity. This is particularly important because the hybridization step of the isothermal amplification method requires a limited temperature range. A strand-displacement strategy can enhance the hybridization efficiency between the probe and target RNA with secondary structure motifs. A short, singlestranded segment within the secondary structure can be used as a toehold for initiating strand displacement. The strategy has been used to establish a highly sensitive isothermal amplification by a combination of a hairpin probe hybridization and stranddisplacement amplification. The hairpin probe is placed on the single-stranded segment of the target RNA's secondary structure to initiate strand displacement. The probe's hybridization cascade provides a template for exponential amplification in two directions by strand-displacement amplification, designated hybridization cascade plus strand-displacement isothermal amplification (HyCaSD). The method requires no reverse transcription step. HyCaSD showed an excellent sensitivity with the limit of detection in the femtomolar (fM) range for synthetic targets as well as viral RNAs. Discrimination between DENV/ ZIKV and JEV/CHIKV was successfully demonstrated using real viruses. Therefore, HyCaSD is a promising platform that can be further developed for diagnostic applications.

Simultaneous Enzyme-Free Detection of Multiple Long Noncoding RNAs in Cancer Cells at Single-Molecule/Particle Level

“…The detection limit can reach 0.031 aM. The sensitivity of this method is much higher than those of previously reported methods for miRNA and viral RNA assays, with 10 orders of magnitude higher than that of the cooperative toehold-mediated strand displacement-based real-time fluorescent method (2 nM), 8 orders of magnitude higher than that of DNA three-way junction-actuated strand displacement-based fluorescent strategy (0.03 nM), 4 orders of magnitude higher than that of hybridization cascade plus strand-displacement isothermal amplification-based fluorescent assay (3.6 fM), and 3 orders of magnitude higher than that of toehold-mediated strand displacement amplification-based electrochemiluminescent biosensor (0.65 fM) . The ultrahigh sensitivity of this method can be ascribed to four factors: (1) lncRNA MALAT1 and lncRNA HOTAIR can specifically recognize the capture probes, initiating the subsequent strand displacement reaction in an orderly manner; (2) the magnetic beads enable direct capture and separation of the reporter units, efficiently eliminating the genetic cross-contamination and reducing the background signal; (3) a large amount of fluorescent molecules per reporter unit greatly amplifies the signal; (4) single-molecule detection offers high signal-to-noise ratio and high sensitivity …”

“…(2) The MB–capture probe–multiple Cy3/Cy5-modified reporter unit complexes can simultaneously recognize lncRNA MALAT1 and lncRNA HOTAIR to generate abundant corresponding fluorescent molecules, greatly amplifying the signals. In contrast to the reported strand displacement amplification strategies with the involvement of hairpin-structure probes, − all the DNA probes used in this research are linear strand DNAs, greatly simplifying the probe design. (3) The introduction of magnetic beads facilitates the separation of lncRNA MALAT1 and lncRNA HOTAIR from the complexes, not only simplifying the experimental operation but also eliminating the genetic cross-contamination in the conventional RNA assays .…”

“…Although these amplification approaches can generate abundant oligonucleotides with high amplification efficiency within minutes, they involve different expensive enzymes such as DNA polymerase, endonucleases, and exonucleases. Alternatively, enzyme-free signal amplification strategies have shown great potential in gene detection. − So far, it has remained a great challenge for the development of a simple and sensitive method for lncRNA assay without the involvement of any nucleic acid amplification and enzymes.…”

Spectrophotometric Detection of the BRCA1 Gene via Exponential Isothermal Amplification and Hybridization Chain Reaction of Surface-Bound Probes

“…To date, DNA isothermal amplification detections have attracted a great deal of research interest because of their simplicity, portability, short assay time, and cost-effectiveness. , Numerous methods including strand displacement amplification (SDA), − rolling circle amplification (RCA), , and nicking enzyme signaling amplification (NESA) have been developed to improve the detection sensitivity in nucleic acid target assay. Hybridization chain reaction (HCR) as one label-free nucleic acid amplification approach has attracted substantial research efforts because it is simple, cost-effective, and enzyme-free.…”

Highly Sensitive Detection of miR-21 through Target-Activated Catalytic Hairpin Assembly of X-Shaped DNA Nanostructures

“…Conventional methods for detecting miRNAs include Northern blotting, microarray hybridization, sequencing, and quantitative reverse transcription polymerase chain reaction (qRT-PCR). , These conventional methods are reliable but usually suffer from limitations, such as sophisticated operation and expensive instrument requirements, poor sensitivity, and complex data analysis. Recently, novel isothermal amplification techniques, including rolling circle amplification (RCA), − strand displacement amplification (SDA), and exponential amplification reaction (EXPAR), have been developed. However, the improved detection sensitivity of isothermal amplification methods may be compromised by the requirement of DNA polymerase or other enzymes and by poor reproducibility.…”