Single Locked Nucleic Acid-enhanced nanopore genetic discrimination of pathogenic serotypes and cancer driver mutations

Abstract

Accurate and rapid detection of single-nucleotide polymorphism (SNP) in pathogenic mutants is crucial for many fields such as food safety regulation and disease diagnostics. Current detection methods involve laborious sample preparations and expensive characterizations. Here, we investigated a single locked nucleic acid (LNA) approach, facilitated by a nanopore singlemolecule sensor, to accurately determine SNPs for detection of Shiga toxin producing Escherichia coli (STEC) serotype O157:H7, and cancer-derived EGFR L858R and KRAS G12D drivermutations. Current LNA applications that require incorporation and optimization of multiple LNA nucleotides. But we found that in the nanopore system, a single LNA introduced in the probe is sufficient to enhance the SNP discrimination capability by over 10-fold, allowing accurate detection of the pathogenic mutant DNA mixed in a large amount of the wildtype DNA. Importantly, the molecular mechanistic study suggests that such a significant improvement is due to the effect of the single-LNA that both stabilizes the fully matched base-pair and destabilizes the mismatched base-pair. This sensitive method, with a simplified, low cost, easy-to-operate LNA design, could be generalized for various applications that need rapid and accurate identification of single-nucleotide variations.

Nanopore-based technologies beyond DNA sequencing

“…These assays involve minimal losses of target molecules during sample preparation while at the same time protecting the nanopore by the selective degradation of background molecules. For example, a biological nanopore-based direct, digital counting of single-nucleotide polymorphic sites marked with locked nucleic acid-synthetic molecules was used for the detection of Shiga toxin-producing Escherichia coli serotype and cancer-derived driver mutations 35,124 . Another approach using solid-state nanopores capitalized on the extremely high specificity of DNA ligase to pull down selected circulating tumour DNA mutations associated with breast cancer genes (that is, ERBB2 and PIK3Ca) in blood samples 34 .…”

Chemistries for DNA Nanotechnology

“…246 Moreover, it has been demonstrated that incorporation of α-L-LNA into DNAzymes can markedly enhanced DNAzyme activity, and recently, LNA proved efficient for detection of single-nucleotide polymorphisms using nanopore technology. 247,248 Likewise, the use of LNA nucleotides proved crucial to Mirkin and co-workers for the creation of superlattices from oligonucleotide-modified gold nanoparticles (AuNPs) of various well-defined shapes. 249 Incorporation of LNA into DNA nanostructures provides a straightforward route to fine-tuning and optimizing nanostructure function.…”

Economics and Mathematical Modeling in Health-Related Research

“…STEC is naturally found in the microbiota of ruminants, which are the most important reservoirs of this zoonotic pathogen (Ballem et al, 2020). Additionally, E. coli O157:H7 is the most important and frequently -978-90-04-51729-5 Downloaded from Brill.com02/13/2023 03:31:30AM via free access isolated serotype of STEC (Siddiqui & Yuan, 2021;Tian et al, 2018). Moreover, the low infectious dose of E. coli O157:H7 may be one of the factors why this serotype is involved in many outbreaks.…”