Research

Fluorescent blinking triggered by Triplet-Triplet energy transfer (TTET)

Cyclooctatetraene (COT) was used as both a triplet acceptor and a photo-stabilizing agent to control and observe the blinking at the single-molecule level. By using DNA as a platform, we demonstrated that triplet blinking of the fluorescent molecule ATTO 647N can be controlled by the collision reaction between COT. The dynamics of biomolecules can be investigated by measuring the blinking. COT and ATTO 647N were attached to the molecular beacon-type probe which allowed us the detection of model biomarker miR-155 at the single-molecule level.

Angew. Chem. Int. Ed., 60, 12941–12948 (2021).

https://resou.osaka-u.ac.jp/en/research/20210414_1

Chem. Commun. 50, 10478–10481 (2014).

Fluorescent blinking triggered by charge transfer in DNA

  By utilizing the transient absorption measurements, we previously showed that charge transfer dynamics in DNA are strongly affected by the DNA sequence. We showed that DNA sequence information including the data on single nucleotide polymorphisms (SNPs) can be read-out by measuring the charge transfer kinetics. However, the measurement requires a significant amount of sample (>1 nmol) and thus cannot be used for diagnosis. We focus on that, by using a fluorophore as a photosensitizer to generate charges on DNA, the charge-separation, charge-transfer, and charge-recombination dynamics in DNA can be monitored as the fluorescence blinking. In this case, the blinking was caused by the successive reduction and re-oxidation cycles of the fluorescent molecule ATTO 655. The τOFF value corresponds to the lifetime of the charge-separated state. Based on the fact that the charge-transfer dynamics in DNA are strongly affected by the DNA sequence, we demonstrated that single-nucleotide differences in DNA that modulate the charge-recombination kinetics can be detected by monitoring the blinking of the fluorescence. We succeeded in the in situ detection of mRNA point mutation on the pathological section.

Chem. Eur. J. 29, e202203552 (2023)

Chem, 8, 3109–3119 (2022).

ChemBioChem, 14, 1430–1433 (2013).

J. Am. Chem. Soc., 133, 15568–15577 (2011).

Fluorescent blinking triggered by redox reaction

  We focused on the control of redox blinking to monitor the microenvironment of the fluorescent molecule. By adding ascorbic acid 2-phosphate (VcP) as a reductant, the triplet state of the fluorescent molecule was converted to the radical anion OFF-state. The duration of the OFF state corresponds to the lifetime of the radical anion of the fluorescent molecule. Here, a bulky oxidant, diethylenetriaminepentaacetic acid iron(III) (FeDTPA), was utilized to regenerate the intact fluorescent molecule at the ground state. Owing to the larger size of FeDTPA, the bimolecular reaction rate between the radical anion of the fluorescent molecule and FeDTPA changed dramatically along with the changes in the microenvironment of the fluorescent molecule. Observation of the redox blinking enabled sensitive detection of subtle conformational changes around the fluorescent molecule caused by single nucleotide alternations in the DNA sequence. Single-molecule detection of target DNA was achieved.

Chem. Eur. J., 24, 6755–6761 (2018).

Angew. Chem. Int. Ed., 56, 15329–15333 (2017).

ChemPhysChem 16, 3590–3594 (2015).

Fluorescent blinking triggered by trans-cis isomerization

  Cyanine dyes such as Cy3 are widely used as fluorescent probes to investigate various biological phenomena. It is known to undergo trans-cis photo-isomerization and successive cis-trans back thermal-isomerization by rotation around the C–C bonds of the poly-methine chain. This cis-trans isomerization causes the blinking of the fluorescence. We focus on the fact that the size of the Cy3 is just about the same as the width of the triple helix. Since the cis-trans isomerization efficiency is considered to be strongly dependent on the steric effects that impact the rotation of the molecule, we hypothesized that Cy3 may exhibit a triple helix-specific blinking. We incorporated Cy3 into DNA double helix, or triple helix conformation and investigated the differences in blinking behavior by Fluorescence Correlation Spectroscopy (FCS). The duration of the on time (τON) and off time (τOFF) of the blinking, which reflect the inverse of the trans-cis photo-isomerization rate and the cis-trans back isomerization rate, respectively, were highest in the triple helix conformation. These results suggest that Cy3 can be used to track the presence of the triple helix conformation at the single-molecule level.

Chem. Commun. 51, 4861–4864 (2015).