α-Hemolysin nanopore sensing of MicroRNA with electrolyte gradients

Abstract

Aberrant microRNA expression profiles have been correlated with a range of complex diseases, including specific types of cancer, hence microRNA species are a promising class of molecular cancer biomarkers. Recently, nanopore technology was proposes as a single-molecule methodology to detect and quantify microRNA molecules without amplification or fluorescent labelling. A duplex of microRNA hybridized with a complementary DNA probe is electrophoretically driven to a nanopore, which can be translocated following duplex unzipping at the channel entrance, which is measured as a transient decrease in nanopore electrical current. Nanopore sensing sensitivity is determined by the occurrence frequency of such resistive current pulses, while the specificity is determined by the probe-analyte interaction. This thesis aims to establish the optimal conditions and limitations of nanopore sensing of cancer-related microRNA species with the biological nanopore α-hemolysin as the sensor element. The fragility of aperture-suspended lipid bilayers is one of the main obstacles for sensing with biological pores, hence we first addressed bilayer stability by laser cutting a thin Teflon film to obtain apertures with a tapered wall profile. Nanopore sensing was then investigated with synthetic miRNA 155, overexpressed in lung cancer, in the presence of a complementary DNA probe. Key parameters of duplex nanopore translocation in conventional symmetrical 1 M KC1 were in agreement with previous work, including a relatively low pulse frequency, allowing quantification of miRNA 155 down to 10 nM. We then systematically investigated the effect of cis/trans KC1 gradients across the nanopore. The resistive pulse frequency increased significantly with the salt gradient, indicative of cation-induced filed enhancement at the pore entrance, but bilayer and pore stability were reduced. At a 0.5 / 4 M gradient, the pulse frequency was ~60 times higher than for symmetrical 1 M KC1 conditions, enabling miRNA quantification down to 100 pM. Additionally, experiments with DNA probes with single and double polynucleotide extensions confirmed the necessity of a double-overhang design under salt gradient conditions, while experiments with NaC1, CsC1 and LiC1 electrolyte gradients suggested that Li addition can extend the duplex unzipping time. Finally, trials were performed with total RNA extracts from clinical samples. Here, bilayer stability was no limitation but pore clogging precluded nanopore sensing, most likely due to longer mRNA species with secondary structure, necessitating further extract processing. Another consideration for nanopore analysis of microRNAs from clinical samples is to minimize the extract resuspension volume, implying the use of miniaturized bilayer recording methodologies.

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Identifiers

ORCID for Philip Williamson: orcid.org/0000-0002-0231-8640

ORCID for Maurits De Planque: orcid.org/0000-0002-8787-0513

ORCID for Hywel Morgan: orcid.org/0000-0003-4850-5676

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