Life science
Medical & healthcare, Drug discovery

Single molecule analysis and diagnosis based on triplet-triplet
energy transfer rate measurements
Department of Material Excitation Chemistry, The Institute of Scientific and Industrial Research (SANKEN)
Associate Professor Kiyohiko Kawai

Researchmap https://researchmap.jp/read0191237

Professor Mamoru Fujitsuka

Researchmap https://researchmap.jp/read0118564

Life science

Abstract
Tracking triplet-triplet energy transfer (TTET) kinetics between
fluorescent molecule and cyclooctatetraene (COT) by monitoring
fluorescence blinking provides a powerful method to access biomolecular dynamics in the time range of μs to ms and detect miRNAs at the single molecule.

▍ Background & Results
The kinetics of photo-induced chemical reaction through its
singlet-excited state S1 can be easily determined by fluorescence
lifetime measurements. However, due to the short lifetime of S1 (10–8seconds), it can only access reactions and dynamic movementsthat occur in a very short time. On the other hand, the triplet excited state T1, which is formed from S1 through intersystem crossing,has a relatively long lifetime (~10–3 s), and thus T1-based kinetics
can be used to investigate reactions and dynamic motions that occur on time scales about 105 times wider than those using S1. TTET
is one of the most useful T1-mediated reactions and has been used
to probe the dynamics of biological macromolecules in the time
range of ~10–3 s. However, T1-based reactions have traditionallybeen measured by transient absorption measurements using laser
flash photolysis, which requires a considerable amount of sample(more than 1 nmole), making it difficult to analyze valuable samples that are difficult to prepare in large quantities. In this study,
we focused on the fact that in single-molecule fluorescence meas-urements, the lifetime of T1 can be measured as the duration of thedark OFF state (τOFF) while the fluorescence is blinking. We haveused COT as both a triplet acceptor and a photo-stabilizer to control and observe blinking at the single molecule level. Using DNA
as a platform, we have shown that triplet blinking of the fluorescentmolecule ATTO 647N can be controlled by the collision reaction
between COT. By attaching COT and ATTO 647N to a molecularbeacon type probe, we were able to detect miR-155, a model biomarker, at the single molecule.

▍ Significance of the research and Future perspective
Förster resonance energy transfer (FRET), a singlet-singlet energy transfer process, occurs inversely proportional to the sixth power of the distance between the two fluorescent molecules. Therefore, FRET occurs over long distances (1-10 nm) and can be used
to study dynamics that induce large distance changes between
two fluorescent molecules, while it is not sensitive to local motion
over small distances. On the other hand, TTET measurements
requires direct Van der Waals contact between 3ATTO 647N* andCOT. Consequently, it can be applied to the dynamics of biomolecules with a small distance change and offers more direct information, which is distinctive from that obtained by FRET. Serving as acomplementary method to FRET, the present method is a versatileand powerful tool for accessing biomolecular dynamics and detecting biomarkers at the single molecule level.

P a t e n tXu, Jie; Fujitsuka, Mamoru; Kawai, Kiyohiko et al. Control of Triplet Blinking Using Cyclooctatetraene to Access the Dynamics of Biomolecules at the Single-Molecule Level. Angewandte Chemie, International Edition. 2021; 60(23): 12941-12948. doi: 10.1002/anie.202101606
UR
L https://www.sanken.osaka-u.ac.jp/labs/mec/kawairesearch.pdf
K e y w o r d single molecule diagnosis, biomarker, fluorescent molecule, triplet state
Tr e a t i s e