Powerful pyrene probes: Two kinds of pyrene-labeled oligonucleotides (HNA- and RNA-skeleton probes) were explored. The enhanced fluorescence intensity in the monomer region and the disappearance of aggregate/excimer emission in duplexes has been successfully used to detect the hybridization of oligonucleotides.
By covalently attaching pyrene chromophores with different linkers onto altritol nucleotides or ribonucleotides, and by varying the number of these pyrene modified altritol nucleotides and ribonucleotides in HNA (hexitol nucleic acid) and RNA, respectively, we have explored the general applicability of pyrene absorbance and especially fluorescence as a probe to monitor RNA hybridization. The results reveal that the backbone of the probes, the number of pyrene units attached and the nature of the tether can all substantially affect the absorbance and fluorescence properties of the probes both in single strand and double strand form. Moreover, the strength of hybridization is also affected. The disappearance of pyrene aggregate/excimer emission and simultaneous increase in monomer emission intensity of the multipyrene-labeled probes has been successfully used to monitor the hybridization of oligonucleotides, including a hairpin structure. Differences in optical response between the HNA- and RNA-skeleton probes upon hybridization indicate that the interaction of pyrene with the nucleobases in both types of duplexes is different.