Hi-fidelity discrimination of isomiRs using G-quadruplex gatekeepers

Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DN...

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Bibliographic Details
Published in:PloS one 2017-11, Vol.12 (11), p.e0188163-e0188163
Main Authors: Seow, Nianjia, Fenati, Renzo A, Connolly, Ashley R, Ellis, Amanda V
Format: Article
Language:English
Subjects:
Biochemistry
Biology and Life Sciences
Biomedical engineering
Computer and Information Sciences
Deoxyribonucleic acid
Design modifications
DNA
DNA probes
Fidelity
Genetic variation
Genomes
Guanine
Hybridization
Hybrids
Ligands
Methods
MicroRNA
MicroRNAs
miRNA
Nuclear electric power generation
Physiological aspects
Potassium
Probes
Research and Analysis Methods
Ribonucleic acid
RNA
RNA-directed DNA polymerase
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Summary:Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0188163