A universal locked nucleic acid-integrated X-shaped DNA probe design for amplified fluorescence detection of single-nucleotide variant

Abstract

Single-nucleotide variant (SNV) is crucial in clinical diagnostics as a useful biomarker, because it is closely associated with many diseases, such as genetic disorders and tumors. In this study, an enzyme-free and selective fluorescence biosensor is proposed for -thalassemia SNV detection based on X-shaped probe, locked nucleic acid (LNA), and toehold-mediated strand-displacement reaction (TMSDR)-assisted recycling technology. On one hand, the LNA-integrated X-shaped probe has high recognition ability for SNV sequence even in the presence of a large amount of wild-type sequence, i.e., the rare SNV can be detected down to 0.01% variant allele frequency (VAF). On the other hand, the introduction of TMSDR-assisted recycling amplification system remarkably improves the sensitivity of the sensor with the detection limit as low as 6 fM mutation gene. Moreover, the designed X-shaped probe is universal because its functionalized oligonucleotides could be decoupled from the target-specific regions. These experimental results demonstrate that this developed method offers high specificity and sensitivity for SNV detection, which might be a potential alternative as an effective mutation detection technology for the detection of -thalassemia SNV and other gene-related diseases.

Principles and Applications of Nucleic Acid Strand Displacement Reactions

“…The latter is displaced by the analyte sequence with the “correct” SNP, leading to an increase in fluorescence. Toehold-mediated strand displacement with two helper DNA strands is used to recycle the analyte, enabling recognition of a single nucleotide variant with a detection limit as low as 6 fM . A similar sensor scheme was combined with an electrochemical sensor platform by Gao et al…”

“…Toehold-mediated strand displacement with two helper DNA strands is used to recycle the analyte, enabling recognition of a single nucleotide variant with a detection limit as low as 6 fM. 346 A similar sensor scheme was combined with an electrochemical sensor platform by Gao et al 347 As the kinetics of strand displacement processes are highly sensitive to the presence of mismatches, this feature can also be utilized for single-base mismatch discrimination. An interesting approach toward SNP genotyping was developed by Khodakov and co-workers who first used PCR of the analyte DNA to generate dsDNA molecules with single-stranded overhangs ("toehold-PCR products").…”

A Ratiometric Fluorescent Bioprobe Based on Carbon Dots and Acridone Derivate for Signal Amplification Detection Exosomal microRNA

“…Furthermore, the selectivity is the other important parameter for detection of miRNAs, because of the presence of complex components in real samples. Supported by some previous studies, including that from our group, the locked nucleic acid (LNA) was incorporated into a DNA strand displacement system, which was able to minimize unwanted reactions and improve the signal-to-noise ratio. , Thus, this ratiometric fluorescent bioprobe possessed high selectivity, even against single base mismatch.…”

Ultraspecific Multiplexed Detection of Low-Abundance Single-Nucleotide Variants by Combining a Masking Tactic with Fluorescent Nanoparticle Counting

“…Many efforts have been devoted to multiplex SNV discrimination and detection, for example, DNA sequencing, , polymerase chain reaction (PCR), − the microarray, − enzyme-assisted methods, − and hybridization-based methods, − where the discrimination readout can be optical, − electrical, mass spectrometric, or electrochemical. ,,,− However, limited approaches can realize multiplex SNV detection with both ultrahigh specificity and sensitivity in a simple manner. To achieve the high discrimination factor and low-abundance detection capability, most available methods require an elaborate and sophisticated probe or primer design, time-consuming procedures, and the tedious amplification step in the workflow. ,,,− In the efforts toward enzyme-free, highly specific SNV discrimination, the emergence of competitive or masking systems sheds lights on the development of highly specific hybridization-based SNV discrimination technologies. ,,,,,, Among the masking or sequester hairpin is an extremely simple masking reagent that enables significantly enhanced SNV discrimination without complicating probe designs for detection, because the hairpin structure will not interfere with the other probes in the test system; in combination with the molecular beacon for signal recognition and signal readout, the question is high discrimination factors (>200) were not consistently achieved for the majority of all tested SNVs. Also, the requirement for dual labeling of the different fluorophore and quencher increases the reagent cost .…”