A mutation-sensitive, multiplexed and amplification-free detection of nucleic acids by stretching single-molecule tandem hairpin probes

Author:

Yang Ya-Jun1,Fu Hang23,Li Xiao-Lu1,Yang Hong-Yu1,Zhou Er-Chi1,Xie Cheng-Yu1,Wu Shu-Wen1,He Fan4,Zhang Yan5,Zhang Xing-Hua1ORCID

Affiliation:

1. Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University , Wuhan 430072, China

2. Wenzhou Institute, University of Chinese Academy of Sciences , Wenzhou , Zhejiang 325011, China

3. School of Physics, University of Chinese Academy of Sciences , Beijing 100049, China

4. Department of Nephrology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan 430030, China

5. Department of Clinical Laboratory, Renmin Hospital of Wuhan University , Wuhan 430072, China

Abstract

Abstract The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G–U or T–G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.

Funder

National Natural Science Foundation of China

China Postdoctoral Science Foundation

Fundamental Research Funds for the Central Universities

Publisher

Oxford University Press (OUP)

Subject

Genetics

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