Surupa Chakraborty, Amity University Kolkata
The past decade saw an explosion in the use of nanopore-based technology for DNA/RNA sequencing where Nanopores can be ideally used as single-molecule sensors. Oxford Nanopore Technologies has developed sequencing technologies – the ION series; a miniature nanopore DNA sequencer called the MinION that allows portable and convenient sequencing of high throughput DNA. A group of scientists redeveloped by using the same technology to detect any target DNA/RNA oligos in a mixture with the help of voltage-driven ion-channel measurements. A unique complementary probe tagged with a bulky Osmium (Osmium tetroxide 2,2’-bipyridine) tag is used in this technique which allows strong hybridization between the probe and target sequence. The osmylated nucleic acid probe, when added to a sample containing its target or complementary sequence, forms a hybrid. When the concentration of the target sequence is equal to or higher than the probe’s concentration, the probe gets hybridized.
OsBp_detect software detects the count of events as compared to the baseline. The presence of bulky Osmium tags on the probes generates multiple counts over the baseline, detected even at a single-digit attomole (amole) range. The ‘silenced’ probes are attributed to the formation of a 1:1 double-stranded (ds) complex that does not fit and cannot traverse the nanopore. These single-molecule sensors are capable of detecting and counting every molecule that traverses the pore. This ready-to-use nanopore-based platform can be rendered beneficial as a diagnostic test for the detection of cell-free tumour DNA (ctDNA) and microRNA (miRNA) and quantitation in body fluids and biopsies.
Consecutive nanopore experiments on the same flow cell, using the probe 2XdmiR122, assured about the sensitivity of this technique in the detection of complementary sequence and also confirmed that the target-probe hybridization results in severely reduced counts, i.e. silencing. The idea of exploiting nanopores as single-molecule sensors have been used previously in many experiments. However, in the era of ubiquitous DNA/RNA sequencing, this novel approach based on ion-channel single-molecule experiments using portable and commercially available nanopore devices from ONT can pave the way to more accurate detection and quantification of highly dilute samples like miRNAs and ctDNA found in bodily fluids.
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Source: Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe. Albert S. W. Kang, Janette G. Bernasconi, William Jack, Anastassia Kanavarioti, (2020), bioRxiv, https://doi.org/10.1101/2020.10.05.327460.
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