Highly selective FRET-aided single-molecule counting of microRNAs labeled by splinted ligation

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Title
Highly selective FRET-aided single-molecule counting of microRNAs labeled by splinted ligation
Author(s)
S Joo; Ui Jin Lee; H Y Son; Moonil Kim; Y M Huh; T G Lee; M Lee
Bibliographic Citation
ACS Sensors, vol. 7, no. 11, pp. 3409-3415
Publication Year
2022
Abstract
MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Forster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.
Keyword
microRNA detectionSingle-molecule countingFRETSplinted ligationSplintR ligase
ISSN
2379-3694
Publisher
Amer Chem Soc
DOI
http://dx.doi.org/10.1021/acssensors.2c01526
Type
Article
Appears in Collections:
Critical Diseases Diagnostics Convergence Research Center > 1. Journal Articles
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