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We developed a technology to measure microRNA (miRNA) copies in serum and tested it on a commercially available combined human serum (H6914 from Sigma-Aldrich). Copies of miR-15b and miR-16, believed to be constant among healthy and diseased individuals, were measured and agreed with the ones reported by Mitchell PS et al. (2008). Cancer biomarkers let7-b, miR-21, miR-141 and miR-375 varied 3,000 to 6,000 copies per 1 microLiter H6914 (5-10 femtomolar (fM)). Detection and quantification of oligos and miRNAs at such low concentration was shown earlier. It is accomplished by repurposing the commercial MinION nanopore platform to conduct single-molecule voltage-driven ion-channel measurements, employing osmium-tagged oligo probes, and using a publicly available algorithm. These miRNAs were quantified in the serum of healthy individuals or cancer patients using novel optimized probes and a detailed protocol that delivers miRNA copies with better than 85% confidence across all concentrations. A linear correlation, the same with healthy and cancer serum samples, is observed between miR-15b copies and RNA concentration in serum. The assay’s simplicity, readiness, sensitivity, and precision advocate for its use as a Laboratory Developed Test (LDT) for disease-screening based on miRNA dysregulation.

Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2′-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact. Earlier reports using OsBp-tagged oligodeoxyribonucleotides demonstrated proof-of-principle during unassisted voltage-driven translocation via either alpha-Hemolysin or a solid-state nanopore. Here we extend this work to RNA oligos and a third nanopore by employing the MinION, a commercially available device from Oxford Nanopore Technologies (ONT). Conductance measurements demonstrate that the MinION visibly discriminates oligoriboadenylates with sequence A15PyA15, where Py is an OsBp-tagged pyrimidine. Such resolution rivals traditional chromatography, suggesting that nanopore devices could be exploited for the characterization of RNA oligos and microRNAs enhanced by selective labeling. The data also reveal marked discrimination between a single pyrimidine and two consecutive pyrimidines in OsBp-tagged AnPyAn and AnPyPyAn. This observation leads to the conjecture that the MinION/OsBp platform senses a 2-nucleotide sequence, in contrast to the reported 5-nucleotide sequence with native nucleic acids. Such improvement in sensing, enabled by the presence of OsBp, may enhance base-calling accuracy in enzyme-assisted DNA/RNA sequencing.

Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies (ONT), and repurposed it to detect any DNA/RNA oligo (target) in a complex mixture by conducting voltage-driven ion-channel measurements. The detection and quantitation of the target is enabled by the use of a unique complementary probe. Using a validated labeling technology, probes are tagged with a bulky Osmium tag (Osmium tetroxide 2,2’-bipyridine), in a way that preserves strong hybridization between probe and target. Intact oligos traverse the MinION’s nanopore relatively quickly compared to the device’s acquisition rate, and exhibit count of events comparable to the baseline. Counts are reported by a publicly available software,OsBp_detect. Due to the presence of the bulky Osmium tag, probes traverse more slowly, produce multiple counts over the baseline, and are even detected at single digit attomole (amole) range. In the presence of the target the probe is “silenced”. Silencing is attributed to a 1:1 double stranded (ds) complex that doesn’t fit and can’t traverse this nanopore. This ready-to-use platform can be tailored as a diagnostic test to meet the requirements for point-of-care cell-free tumor DNA (ctDNA) and microRNA (miRNA) detection and quantitation in body fluids.

Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects in-vitro or in-vivo may be partially related to low purity and the presence of shorter sequences.

Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. 'Base calling' becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.