A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening

Abstract

Background Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics. Methods Nineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance. Results The data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7–8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3–97.5%) and 92.2% (95% CI, 94.7–100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6–99.7%) and 92% sensitivity (95% CI, 86.1–95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%–99.1%) and 62.5% sensitivity (95% CI, 35.8%–83.7%) with AUC of 0.79 for precancerous lesions cfDNA. Conclusions The XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

A highly sensitive XNA-based RT-qPCR assay for the identification of ALK, RET, and ROS1 fusions in lung cancer

“…We harnessed our existing XNA technology, previously employed for Single Nucleotide Polymorphism (SNP) detection in genomic DNA samples [31], to design assays for the identification of gene fusion transcripts. Specifically, we formulated panels targeting 28 well-known fusion transcripts, encompassing over 90% of ALK, RET, or ROS1 fusion transcripts [38].…”

“…In multiplex PCR-based assays, off-target amplification is a common issue [34]. Particularly, in the presence of a substantial background of wild-type sequences, false positives in mutation or fusion detection tend to increase, primarily due to the promiscuous binding of primers and probes to sequences that closely resemble the target [31]. To mitigate non-specific amplification in the presence of abundant wild-type backgrounds, we assessed the detection efficiency of each Qfusion TM assay by employing 10-fold serial dilutions of ALK, RET, or ROS1 wild-type RNA transcripts.…”

“…The attachment of XNA to its designated sequence obstructs the extension of the DNA strand by DNA polymerase. In cases where there is a mismatch in the target site sequence, the XNA-DNA duplex lacks stability, permitting the extension of the strand by DNA polymerase [31,32].…”

A novel XNA-based Luminex assay to detect UBA1 somatic mutations associated with VEXAS syndrome

“…This amalgamation offers a straightforward, robust, and highly sensitive approach for detecting multiple mutation targets in a single run. The XNA clamping technique effectively curtails wild-type amplification and enriches mutant sequence amplification during PCR, enabling the detection of mutations at a very low variant allele frequency (VAF) of 0.1–0.5% [ 12 ]. Leveraging the high fidelity of Taq DNA ligase, which demonstrates remarkable discrimination for single-base mismatches on the 3′-side [ 13 ], the assay has further increased sensitivity to 0.1% VAF and high specificity for mutations even within the same codon.…”

Rapid Detection of SARS-CoV-2 Variants by Molecular Clamping Technology Based RT-qPCR

“…This appealing feature allows for the highly speci c clamping of the XNA molecule onto the targeted sequence (usually the wildtype, WT) to block WT ampli cation, thus minimizing WT background in RT-qPCR and selectively enhancing the signal of the mutant. Robust XNA based assays have been extensively used for in vitro diagnostic assays for detecting rare cancer-associated gene mutations 34 . Based on their molecular properties and prior utility in cancer mutation detection, XNAs represent an ideal tool for developing highly sensitive RT-qPCR assays for detecting SARS-CoV-2 mutations.…”