Abstract
We report a universal smart probe (SP) that is capable of detecting several homologous let-7 microRNAs (miRNAs). While the SP is complementary to let-7a, and therefore, strongly binds to this target, due to sequence homology, the SP also has equal propensity to non-specifically hybridize with let-7b and let-7c, which are homologous to let-7a. The fluorescence signal of the SP was switched off in the absence of any homologous member target, but the signal was switched on when any of the three homologous members was present. With the assistance of nucleic acid blockers (NABs), this SP system can discriminate between homologous miRNAs. We show that the SP can discriminate between let-7a and the other two sequences by using linear NABs (LNABs) to block non-specific interactions between the SP and these sequences. We also found that LNABs used do not cross-react with the let-7a target due to the low LNABs:SP molar ratio of 6:1 used. Overall, this SP represents a universal probe for the recognition of a homologous miRNA family. The assay is sensitive, providing a detection limit of 6 fmol. The approach is simple, fast, usable at room temperature, and represents a general platform for the in vitro detection of homologous microRNAs by a single fluorescent hairpin probe.
Highlights
Smart probes (SPs) are singly-labelled fluorescent probes used for the sequence-specific recognition of nucleic acid targets [1,2,3,4,5]
Unlike other methods that have so far been used for miRNA detection, homogeneous methods based on the smart probe (SP)/nucleic acid blockers (NABs) system can offer good sensitivity, while presenting excellent discrimination between the miRNA target of interest and similar mismatch sequences, including single nucleotide polymorphism (SNP)
The label shown for each nucleic acid in the right column of the table is hereby adopted for the rest of this paper
Summary
Smart probes (SPs) are singly-labelled fluorescent probes used for the sequence-specific recognition of nucleic acid targets [1,2,3,4,5] They possess stem-loop, hairpin conformation, with a fluorescent dye on one end of the oligonucleotide sequence and guanine bases on the other. If a nucleic acid sequence has a single-base mutation in it, the SP can differentiate between this mismatch and the target sequence by providing a relatively lower fluorescence signal compared to that of the perfect target, which indicates a less stable hybrid between the SP and the mismatch sequence.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
