Detection of nucleic acids is crucial to the study of their basic properties and consequently to applying this knowledge to the determination of pathologies such as cancer. In this work, our goal is to determine new trends for creating diagnostic tools for cancer driver mutations. Herein, we study a library of natural and modified oligonucleotide duplexes by a combination of optical and theoretical methods. We report a profound effect of additives on the duplexes, including nucleic acids as an active crowder. Unpredictably and inconsistent with DNA+LNA/RNA duplexes, locked nucleic acids contribute poorly to mismatch discrimination in the DNA+LNA/DNA duplexes. We develop a theoretical framework that explains poor mismatch discrimination in KRAS oncogene. We implement our findings in a bead-bait genotyping assay to detect mutated human cancer RNA. The performance of rationally designed probes in this assay is superior to the LNA-primer polymerase chain reaction, and it agrees with sequencing data.
© 2020. The Author(s).