Exposure of DNA to chemicals can result in the formation of DNA adducts, a molecular initiating event in genotoxin-induced carcinogenesis. O-6-Methylguanine (O-6-MeG) is a highly mutagenic DNA adduct that forms in human genomic DNA upon reaction with methylating agents of dietary, environmental, or endogenous origin. In this work, we report the design and synthesis of novel non-natural nucleoside analogues 1'-beta-[1-naphtho[2,3-d]imidazol-2(3H)-one)]-2'-deoxy-D-ribofuranose and 1'-beta-[1-naphtho [2,3-d]imidazole]-2'-deoxy-D-ribofuranose and their use for quantifying O-6-MeG within mutational hotspots of the human KRAS gene. The novel nucleoside analogues were incorporated into oligonucleotides conjugated to gold nanoparticles to comprise a DNA hybridization probe system for detecting O-6-MeG in a sequence-specific manner on the basis of colorimetric readout of the nanoparticles. The concept described herein is unique in utilizing new nucleoside analogues with elongated hydrophobic surfaces to successfully measure in-gene abundance of O-6-MeG in mixtures with competing unmodified DNA.