Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection.

MicroRNAs (miRNAs) are a class of noncoding RNAs that are being explored as a new type of disease biomarkers. The nanopore single-molecule sensor offers a potential noninvasive tool to detect miRNAs for diagnostics and prognosis applications. However, one of the challenges that limits its clinical applications is the presence of a large variety of nontarget nucleic acids in the biofluid extracts. Upon interacting with the nanopore, nontarget nucleic acids produce "contaminative" nanopore signals that interfere with target miRNA discrimination, thus severely lowering the accuracy in target miRNA detection. We have reported a novel method that utilizes a designed polycationic peptide-PNA probe to specifically guide the target miRNA migration toward the nanopore, whereas any nontarget nucleic acids without the probe bound is rejected by the nanopore. Consequently, nontarget species are driven away from the nanopore and only the target miRNA can be detected at low concentration. This method is also able to discriminate miRNAs with single-nucleotide difference by using PNA to capture miRNA. Considering the significance and impact of this substantial advance for the future miRNA detection in biofluid samples, we prepared this detailed protocol, by which the readers can view the experimental procedure, data analysis, and resulting explanation.

Nanolock-Nanopore Facilitated Digital Diagnostics of Cancer Driver Mutation in Tumor Tissue.

Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock-nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock-nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.

Cerebral arterial fenestrations.

Arterial fenestrations are an anatomic variant with indeterminate significance. Given the controversy surrounding fenestrations we sought their prevalence within our practice along with their association with other cerebrovascular anomalies. We retrospectively reviewed 10,927 patients undergoing digital subtraction angiography between 1992 and 2011. Dictated reports were searched for the terms "fenestration" or "fenestrated" with images reviewed for relevance, yielding 228 unique cases. A Medline database search from February 1964 to January 2013 generated 304 citations, 127 cases of which were selected for analysis. Cerebral arterial fenestrations were identified in 228 patients (2.1%). At least one aneurysm was noted in 60.5% of patients, with an aneurysm arising from the fenestration in 19.6% of patients. Aneurysmal subarachnoid hemorrhage or non-aneurysmal subarachnoid hemorrhage were present in 60.1% and 15.8%, respectively. For the subset of patients with an aneurysm arising directly from a fenestration relative to those patients with an aneurysm not immediately associated with a fenestration, the prevalence of aneurysmal subarachnoid hemorrhage was 66.7% vs. 58.6% (p = 0.58). Fenestrations were more often within the posterior circulation (73.2%) than the anterior circulation (24.6%), though there was no difference in the prevalence of aneurysms within these groups (61.1% vs. 60.7%, p = 1.0). Cerebral arterial fenestrations are an anatomic variant more often manifesting at the anterior communicating arterial complex and basilar artery and with no definite pathological relationship with aneurysms.