Single nucleotide polymorphism (SNP) discriminations by nanopore sensing

Single Nucleotide Polymorphisms (SNPs) are a common type of nucleotide alterations across the genome. A rapid but accurate detection of individual or SNP panels can lead to the right and in-time treatments which possibly save lives. In one of our studies, nanopore is introduced to rapidly detect BRAF 1799 T-A mutation (V600E), with the help of an Ap-dA cross-link right at the mutation site. These sequence-specific crosslinks are formed upon strong covalent interactions between probe based abasic sites (Ap) and target based deoxy-adenosine (dA) residues. Duplexes stabilized by the crosslink complexes create indefinite blocking signatures when captured in the nanopore, creating a high contrast compared to the "spike-like" translocations events produced by the un-crosslinked and wildtype duplexes. Those consistent blocking events couldn't be resolved unless an inverted voltage is applied. In a 1:1 BRAF mutant-wildtype mixture, the nanopore can successfully discriminate between the two sequences in a quantitative manner. In summary, nanopore paired with sequence-specific crosslink can detect a specific type of SNP with a high contrast manner. In another study, nanopore sensing is modified to be capable of detections with multiple SNPs in a single detection mix. To achieve this, an RNA homopolymer barcode is integrated into the probe sequence so nanopore can read out a distinctive level signature when the target-probe duplex is de-hybridizing through the pore. Since different RNA homopolymers (e.g. Poly rA and Poly rC) can generate signature levels distinctive from each other and other DNA sequences, they can be applied to generate characteristic patterns that simultaneously highlight multiple SNPs in the mixture. In this study, we assigned two different RNA barcodes (Poly rA and Poly rC) to label KRAS G12D and Tp53 R172H SNPs (both T[right arrow]A mutations) in the solution. During nanopore readout, the KRAS G12D containing duplex generates a "downward" step pattern but Tp53 R172H always has an "upward" step pattern, the high contrast between those two patterns makes recognition easy enough with naked eyes, and further statistical analysis is unnecessary. Theoretically, at least four different barcodes can be implemented at the same time, furthermore, the length of the barcode can also affect the barcode pattern. Thus, in theory, a panel of more than 10 SNPs can be identified simultaneously.Includes bibliographical reference

Hepatic Phospholipid Remodeling Modulates Insulin Sensitivity and Systemic Metabolism

Abstract The liver plays a central role in regulating glucose and lipid metabolism. Aberrant insulin action in the liver is a major driver of selective insulin resistance, in which insulin fails to suppress glucose production but continues to activate lipogenesis in the liver, resulting in hyperglycemia and hypertriglyceridemia. The underlying mechanisms of selective insulin resistance are not fully understood. Here It is shown that hepatic membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) regulates insulin signaling and systemic glucose and lipid metabolism. Hyperinsulinemia induced by high‐fat diet (HFD) feeding augments hepatic Lpcat3 expression and membrane unsaturation. Loss of Lpcat3 in the liver improves insulin resistance and blunts lipogenesis in both HFD‐fed and genetic ob/ob mouse models. Mechanistically, Lpcat3 deficiency directly facilitates insulin receptor endocytosis, signal transduction, and hepatic glucose production suppression and indirectly enhances fibroblast growth factor 21 (FGF21) secretion, energy expenditure, and glucose uptake in adipose tissue. These findings identify hepatic LPCAT3 and membrane phospholipid composition as a novel regulator of insulin sensitivity and provide insights into the pathogenesis of selective insulin resistance

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 <i>BRAF</i> 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 <i>BRAF</i> 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