FRET-based dual-labeled oligonucleotide probes detect target DNA by probing a minor groove environment

Abstract

A novel amino-modifier ESF nucleoside ^AM37zA (1) containing a trifluoroacetyl (TFA)-protected amino group is designed for the functionalization of ODN probes after oligonucleotide synthesis. ^AM37zA (1) showed remarkable solvatochromicity and ODN probes containing deprotected ^AM37zA [ODN(^N37zA)] discriminated single base alteration in target DNA based on changes in the fluorescence wavelength and intensity. Using ^AM37zA (1), a FRET-based dual-labeled ODN probe, ODN(^F37zA), which contains two fluorophores at the major and minor groove sides of DNA, has been synthesized. The ODN(^F37zA) incorporated with fluorescein by post-synthetic modification discriminated the target DNA sequence by a distinct change in the fluorescence intensity (S/N ratio of 20 : 1). Thus, an amino-modifier, ^AM37zA, and a FRET-based ODN probe, ODN(^F37zA), are valuable for monitoring the microenvironment around nucleic acids and can act as powerful tools for examining the microstructures of DNA/RNA, including gene detection.