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

• Published in: Sensors and actuators. B, Chemical Chemical, 2017-03, Vol.241, p.123-128
• Main Authors: Wu, Fang, Chen, Mei, Lan, Jianming, Xia, Yaokun, Liu, Mengmeng, He, Wenhui, Li, Chunyan, Chen, Xiaosong, Chen, Jinghua
• Format: Article
• Language: English
• Subjects: Amplification; Biomarkers; Biosensors; Deoxyribonucleic acid; Design for recycling; Diseases; DNA; Fluorescence; Fluorescence detection; Locked nucleic acid; Mutations; Oligonucleotides; Recycling; Sensitivity; Sensors; Single-nucleotide variant; Toehold-mediated strand-displacement reaction; X-shaped DNA probe; β-thalassemia
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2016.10.066

•An enzyme-free and selective fluorescence biosensor for β-thalassemia SNV detection is proposed based on X-shaped probe.•Locked nucleic acid integrated in X-shaped DNA probe improves the stability and specificity of the sensor.•Signal amplification of the fluorescence biosensor lies in the toehold-mediated strand-displacement reaction assisted recycling. 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 β-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 β-thalassemia SNV and other gene-related diseases.