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Nicking-enhanced rolling circle amplification for sensitive fluorescent detection of cancer-related microRNAs
- Source :
- Analytical and Bioanalytical Chemistry. 410:6819-6826
- Publication Year :
- 2018
- Publisher :
- Springer Science and Business Media LLC, 2018.
-
Abstract
- In this study, a biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) was proposed for the highly sensitive detection of cancer-related let-7a microRNA (miRNA). The sensing system consists of a padlock probe (PP), which contains a target recognition sequence and two binding sites for nicking endonuclease (NEase), and molecular beacon (MB) as reporting molecule. Upon hybridization with let-7a, the PP can be circularized by ligase. Then, the miRNA acted as polymerization primer to initiate rolling circle amplification (RCA). With the assistance of NEase, RCA products can be nicked on the cyclized PP and are displaced during the subsequent duplication process, generating numerous nicked fragments (NFs). These NFs not only induce another RCA reaction but also open the molecular beacons (MBs) via hybridization, leading to significantly amplified fluorescence signal. Under the optimized conditions, this method exhibits high sensitivity toward target miRNA let-7a with a detection limit of as low as 10 pM, a dynamic range of three orders of magnitude is achieved, and its family member is easily distinguished even with only one mismatched base. Meanwhile, it displays good recovery and satisfactory reproducibility in fetal bovine serum (FBS). Therefore, these merits endow the newly proposed N-RCA strategy with powerful implications for miRNA detection. Graphical abstract A biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) for the highly sensitive detection of cancer-related let-7a microRNA.
- Subjects :
- 0301 basic medicine
010402 general chemistry
01 natural sciences
Biochemistry
Analytical Chemistry
03 medical and health sciences
Endonuclease
Recognition sequence
Limit of Detection
Molecular beacon
Cell Line, Tumor
Neoplasms
Humans
chemistry.chemical_classification
Detection limit
DNA ligase
biology
Reproducibility of Results
0104 chemical sciences
Native Polyacrylamide Gel Electrophoresis
MicroRNAs
Spectrometry, Fluorescence
030104 developmental biology
chemistry
Rolling circle replication
biology.protein
Biophysics
Primer (molecular biology)
Nucleic Acid Amplification Techniques
Biosensor
Subjects
Details
- ISSN :
- 16182650 and 16182642
- Volume :
- 410
- Database :
- OpenAIRE
- Journal :
- Analytical and Bioanalytical Chemistry
- Accession number :
- edsair.doi.dedup.....996df55c139b5f68e606fcae9a882686