Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe

Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies, 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 does not fit and cannot 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.

: Samples of tsv files, obtained by running the OsBp_detect software on fast-5 files.
Left, sample probe T8(RNA); right, sample is a mixture of d(CT) 10   molecules bumping at the pore aperture, without traversing the pore and they are not counted when selecting "All Ir/Io < 0.6" (see Fig. S1, and Experimental Section).   Right, Consecutive nanopore experiments using the same device, the same duration, and voltage. First, we tested a sample made of equimolar amounts of the intact BJ1, BJ2, BJ3, BJ4 (BJs) at a total concentration of 5µM, and then we tested a sample of the osmylated probe BJ4 also at 5 µM. The counts produced by the intact DNA oligos are 1/10 of the counts produced by the DNA probe BJ4 under identical conditions. We attribute the different translocation profiles observed for probe BJ4 to an aged flow cell (left) and to a new flow cell (right).  table). Left profile the same as in Figure 3c, repeated here for direct comparison with the right profile. The difference in these two cases is that, due to the added A 15 -tail, miRNA21-A 15 elutes couple of min later compared to miRNA21.
Bottom profile, one sample only, the 1:1 mixture of miRNA 21 with dmiR21 at two different wavelengths to show that only the probe exhibits absorbance at 312nm, whereas native nucleic acids, like miRNA21, exhibit negligible absorbance at 312nm. Two HPLC peaks are consistent with no hybridization.
Samples in about 90% ONT buffer as the sample solvent. HPLC profiles obtained with HPLC method B (Experimental Section). HPLC profile of the mixture sample matches closely the sum of the HPLC profiles of the two components, providing evidence for no detectable hybridization in these two cases. in order to compare directly with HPLC profile (green trace) to the right. Right: HPLC profiles of three samples in 15% serum -85% ONT buffer: miRNA140 (blue trace) 2min incubation before analysis, 100nt RNA (red trace) 30min incubation before analysis. The longer incubation is why the degradation of 100nt RNA appears more severe compared to the degradation of miRNA140. Mixture of dmiR21(OMe)(OsBp): miRNA21=1:2 in about 5% water-95% ONT buffer (left, red trace) and the same mixture in 15% serum -85% ONT buffer (right, green trace). The HPLC profiles (red trace in left profile and green trace in right profile) appear comparable suggesting that the hybrid suffers insignificant degradation in 15% serum -85% ONT buffer. HPLC method B used for these analyses (see Fig. S10 and Experimental Section).

Figure S10: HPLC profiles of the 2 intact complements of primerM13for(-20) and primerM13for(-41) shown at 260 nm, and their corresponding T-osmylated derivatives
shown at 272nm and 312nm using HPLC method B (see Experimental Section). Analysis of oligos in water as the sample solvent. Briefly HPLC Method B is using the DNA Pac PA200 HPLC column from ThermoFisher Scientific at the 2x250mm configuration with 0.45mL/min flow and 15ºC column compartment. Solvents are aqueous pH 8.0±0.2 mobile phases A (MPA) and mobile phase B (MPB) with 25mM TRIS.HCL buffer; MPB is 1.5 M NaCl. Initial conditions are 90% MPA -10% MPB, and the gradient is from 10% to 50% MPB in 20 min. The total analysis time including column equilibration is 30min. T-osmylation of these oligos was conducted using protocol o (see Experimental Section). Right profile shows an atypical, but confirmed result, namely an osmylated conjugate that elutes later compared to the parent intact nucleic acid. properties with numerous counts at a relatively low probe load (Fig. 4d). A nanopore experiment conducted with BJ2 AT(OMe) at comparable conditions was not conclusive. Since the only difference between the two probes is that the 3'-end on one is the 5'-end on the other, it is not clear whether or not BJ2 TA(OMe) with the Ts at the 5'-end and the As at the 3'-end is a superior probe compared to BJ2 AT(OMe) with the Ts at the 3'-end and the As at the 5'-end.  OsBp, earlier process where bipy was dissolved after adding OsO 4 (see Experimental Section). Materials in water for analysis, and analysis was done using HPLC method B (see Fig. S10 and Experimental Section).  (see table   for sequences and for protocols). Materials were in water for analysis and analysis was done using HPLC method B (see Fig. S10 and Experimental Section). OsBp (earlier process, bipy not dissolved prior to OsO 4 addition. Bottom, probe 140EXT(mU) intact and its osmylation products with the two different protocols; prep2, using protocol a, 45min with 2.63mM OsBp, and prep1 protocol b, 30min with 2.63mM OsBp. Materials in water for analysis, and analysis was done using HPLC method B (see Fig. S10 and Experimental Section).

No of U in the sequence
Osmylation of oligos that are missing T bases per protocol c. Oligos with U (blue) or U in a sequence with either all bases or only 50% of the bases 2'-OMe (red)