Seow Nianjia, Fenati Renzo A, Connolly Ashley R, Ellis Amanda V
Flinders Centre for Nanoscale Science and Technology, Flinders University, Bedford Park, SA, Australia.
School of Chemical and Biomedical Engineering, The University of Melbourne, Parkville, Victoria, Australia.
PLoS One. 2017 Nov 16;12(11):e0188163. doi: 10.1371/journal.pone.0188163. eCollection 2017.
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.
核心微小RNA（miRNA）序列以称为异源miRNA的变体群体形式存在，这些变体由不同长度和核苷酸组成。特别是，miRNA的短序列使得单碱基异源miRNA错配很难被区分。当DNA探针与miRNA靶标的杂交是主要或初始检测模式时，常常会出现非特异性杂交。这些错误会在随后的扩增步骤中进一步加剧。在此，我们展示了用聚鸟嘌呤（PG）序列修饰的DNA探针的设计，这些序列被诱导形成G-四链体（G4），用于从单碱基错配的异源miRNA中高保真地识别miRNA核心靶序列。我们证明，与未修饰的探针相比，G4修饰探针中的这种G4“守门”功能能够使互补的核心miRNA靶转录本进行更严格的杂交，同时限制非特异性杂交。即使在探针-靶标杂交体进一步逆转录酶延伸后，G4修饰探针相对于未修饰探针的这种增强的区分能力仍然保持。酶促延伸还提高了读数的清晰度和灵敏度，并允许通过错配的相对位置区分不同的异源miRNA。
