Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with Peptide Nucleic Acid Hybridization Probes

Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a hybridization-based assay, in which the excess of the DNA hybridization probes is separated from the miRNA-probe hybrids, and the hybrids are separated from each other in gel-free capillary electrophoresis (CE) using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE running buffer and peptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNA hybridization probes and requires no SSB in the CE running buffer. PNA probes are electrically neutral, while PNA-miRNA hybrids are negatively charged, and this difference in charge can be a basis for separation of the hybrids from the probes. In this proof-of-principle work, we first experimentally confirmed that the PNA-RNA hybrid was separable from the excess of the PNA probe without SSB in the running buffer, resulting in a near 10 min time window, which would allow, theoretically, separation of up to 30 hybrids. Then, we adapted to PNA-RNA hybrids our previously developed theoretical model for predicting hybrid mobilities. The calculation performed with the modified theoretical model indicated that PNA-RNA hybrids of slightly different lengths could be separated from each other without drag tags. Accordingly, we designed a simple experimental model capable of confirming: (i) separation of tag-free hybrids of different lengths and (ii) separation of same-length hybrids due to a drag tag on the PNA probe. The experimental model included three miRNAs: 20-nt miR-147a, 20-nt miR-378g, and 22-nt miR-21. The three complementary PNA probes had lengths matching those of the corresponding target miRNAs. The probe for miR-147a had a short five-amino-acid drag tag; the other two had no drag tags. We were able to achieve baseline separation of the three hybrids from each other. The LOQ of 14 pM along with the high accuracy (recovery >90%) and precision (RSD ≈ 10%) of the assay at picomolar target concentrations suggest that PNA-facilitated DQAMmiR could potentially support practical miRNA analysis of clinical samples.