A Universal Strategy for Enhancing the Circulating miRNAs’ Detection Performance of Rolling Circle Amplification by Using a Dual-Terminal Stem-Loop Padlock

Abstract

Rolling circle amplification (RCA) is one of the most promising nucleic acid detection technologies and has been widely used in the molecular diagnosis of disease. Padlock probes are often used to form circular templates, which are the core of RCA. However, RCA often suffers from insufficient specificity and sensitivity. Here we report a reconstruction strategy for conventional padlock probes to promote their overall performance in nucleic acid detection while maintaining probe functions uncompromised. When two rationally designed stem-loops were strategically placed at the two terminals of linear padlock probes, the specificity of target recognition was enhanced and the negative signal was significantly delayed. Our design achieved the best single-base discrimination compared with other structures and over a 1000-fold higher sensitivity than that of the conventional padlock probe, validating the effectiveness of this reconstruction. In addition, the underlying mechanisms of our design were elucidated through molecular dynamics simulations, and the versatility was validated with longer and shorter padlocks targeting the same target, as well as five additional targets (four miRNAs and dengue virus – 2 RNA mimic (DENV-2)). Finally, clinical applicability in multiplex detection was demonstrated by testing real plasma samples. Our exploration of the structures of nucleic acids provided another perspective for developing high-performance detection systems, improving the efficacy of practical detection strategies, and advancing clinical diagnostic research.

Endogenous Enzyme-Driven Amplified DNA Nanocage Probe for Selective and Sensitive Imaging of Mature MicroRNAs in Living Cancer Cells

“…The operation procession, such as heating and RNA extraction, hinders the use of RT-qPCR for in situ intracellular miRNA analysis in living cells. Alternatively, benefiting from the in situ real-time analysis ability of fluorescence imaging, as well as the good biocompatibility and high programmability of DNA materials, − many DNA-based fluorescence probes were developed for intracellular miRNA imaging. These probes based on spherical nucleic acid, , molecular beacons (MB), hybridization chain reaction (HCR), and catalytic hairpin assembly (CHA) can respond to miRNAs due to the sequence complementarity between probes and miRNAs.…”

CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output

“…RNAs, as genetic massagers in biological cells, play an irreplaceable role in gene coding and regulation across almost all life processes. , Its great value in disease diagnosis and prognosis assessment has also attracted considerable interest in recent years . For instance, bacterial 16S rRNA (rRNA), which is highly conserved between different bacterial species and relatively abundant in viable cells, serves as an ideal target in species-specific pathogen identification. − Noncoding RNAs, including MicroRNAs and long noncoding RNA (lncRNA), are another important kind of RNA that participate in life activities as “organizers” and regulatory molecules, and their aberrant expression has become an important diagnostic and prognostic biomarker for malignant tumors, cardiovascular diseases, and nervous system diseases. , Although traditional methods, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Northern blots, and microarray analysis, have been well established for RNA detection, the involvement of tedious process, dedicated equipment, and trained personnel largely limited their applications to centralized laboratories. , Isothermal amplification techniques, including nucleic acid sequence-based amplification (NESBA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), avoid complex thermal cycling and shorten the detection time. − However, their applications are still restricted by the sophisticated primer design and the risk of aerosol contamination. Therefore, a rapid, simple, and sensitive method for RNA detection remains an urgent need.…”