Quencher-free molecular beacon tethering 7-hydroxycoumarin detects targets through protonation/deprotonation.
In this study, we synthesized a simple but efficient quencher-free molecular beacon tethering 7-hydroxycoumarin on D-threoninol based on its pK(a) change. The pK(a) of 7-hydroxycoumarin in a single strand was determined as 8.8, whereas that intercalated in the duplex was over 10. This large pK(a) shift (more than 1.2) upon hybridization could be attributed to the anionic and hydrophobic microenvironment inside the DNA duplex. Because 7-hydroxycoumarin quenches its fluorescence upon protonation, the emission intensity of the duplex at pH 8.5 was 1/15 that of the single strand. We applied this quenching mechanism to the preparation of a quencher-free molecular beacon by introducing the dye into the middle of the stem part. In the absence of the target, the stem region formed a duplex and fluorescence was quenched. However, when the target was added, the molecular beacon opened and the dye was deprotonated. As a result, the emission intensity of the molecular beacon with the target was 10 times higher than that without the target. Accordingly, a quencher-free molecular beacon utilizing the pK(a) change was successfully developed.
Graduate School of Engineering, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8603, Japan.
Hiromu
H
Kashida
Kyohei
K
Yamaguchi
Yuichi
Y
Hara
Hiroyuki
H
Asanuma
D000605:Amino Alcohols / Q000737:chemistry; D002072:Butylene Glycols / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007364:Intercalating Agents / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D011522:Protons; D014468:Umbelliferones / Q000737:chemistry*
D000605:Amino Alcohols; D002072:Butylene Glycols; D007364:Intercalating Agents; D011522:Protons; D014468:Umbelliferones; C532652:threoninol; C031477:7-hydroxycoumarin; D004247:DNA