Cationic conjugated polyelectrolyte/molecular beacon complex for sensitive, sequence-specific, real-time DNA detection.
A new fluorescence method has been developed for DNA detection at room temperature in a sensitive, selective, economical, and real-time manner that interfaces the superiority of a molecular beacon in mismatch discrimination with the light-harvesting property of water-soluble conjugated polyelectrolytes. The probe solution contains a cationic conjugated polyelectrolyte (PFP-NMe3+), a molecular beacon with a five base pairs double-stranded stem labeled at the 5'-terminus with fluorescein (DNA P-Fl), and ethidium bromide (EB, a specific intercalator of dsDNA). The electrostatic interactions between DNA P-Fl and PFP-NMe3+ keep them in close proximity, facilitating the fluorescence resonance energy transfer (FRET) from PFP-NMe3+ to fluorescein. Upon adding a complementary strand to the probe solution, the conformation of DNA P-Fl transits into dsDNA followed by the intercalation of EB into the grooves. Two-step FRET, from PFP-NMe3+ to DNA P-Fl (FRET-1), followed by FRET from DNA P-Fl to EB (FRET-2) takes place. In view of the observed fluorescein or EB emission changes, DNA can be detected in aqueous solution. Because the base mismatch in target DNA inhibits the transition of DNA P-Fl from the stem-loop to duplex structure, single nucleotide mismatch can be clearly detected.
Feng
Xuli
X
Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, PR China.
Duan
Xinrui
X
Liu
Libin
L
An
Lingling
L
Feng
Fude
F
Wang
Shu
S
D002412:Cations; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004573:Electrolytes / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D002412:Cations; D004573:Electrolytes; D004247:DNA