Nucleic Acid Hybridization‐Based Noise Suppression for Ultraselective Multiplexed Amplification of Mutant Variants

Abstract The analysis of mutant nucleic acid (NA) variants can provide crucial clinical and biological insights for many diseases. Yet, existing analysis techniques are generally constrained by nonspecific “noise” signals from excessive wildtype background sequences, especially under rapid isothermal multiplexed target amplification conditions. Herein, the molecular hybridization chemistry between NA bases is manipulated to suppress noise signals and achieve ultraselective multiplexed detection of cancer gene fusion NA variants. Firstly, modified locked NA (LNA) bases are rationally introduced into oligonucleotide sequences as designed “locker probes” for high affinity hybridization to wildtype sequences, leading to enrichment of mutant variants for multiplexed isothermal amplification. Secondly, locker probes are coupled with a customized “proximity‐programmed” (SERS) readout which allows precise control of hybridization‐based plasmonic signaling to specifically detect multiple target amplicons within a single reaction. Moreover, the use of triple bond Raman reporters endows NA noise signal‐free quantification in the Raman silent region (≈1800−2600 cm⁻¹). With this dual molecular hybridization‐based strategy, ultraselective multiplexed detection of gene fusion NA variants in cancer cellular models is actualized with successful noise suppression of native wildtype sequences. The distinct benefits of isothermal NA amplification and SERS multiplexing ability are simultaneously harnessed.