Biophysics and the Challenges of Emerging Threats Paperback / softback

Edited by Joseph Puglisi

Part of the NATO Science for Peace and Security Series B: Physics and Biophysics series

Paperback / softback

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Description

Single-molecule techniques eliminate ensemble averaging, thus revealing transient or rare species in heterogeneous systems [1–3].

These approaches have been employed to probe myriad biological phenomena, including protein and RNA folding [4–6], enzyme kinetics [7, 8], and even protein biosynthesis [1, 9, 10].

In particular, immobilization-based fluorescence te- niques such as total internal reflection fluorescence microscopy (TIRF-M) have recently allowed for the observation of multiple events on the millis- onds to seconds timescale [11–13].

Single-molecule fluorescence methods are challenged by the instability of single fluorophores.

The organic fluorophores commonly employed in single-molecule studies of biological systems display fast photobleaching, intensity fluctuations on the millisecond timescale (blinking), or both.

These phenomena limit observation time and complicate the interpretation of fl- rescence fluctuations [14, 15].

Molecular oxygen (O) modulates dye stability. Triplet O efficiently 2 2 quenches dye triplet states responsible for blinking.

This results in the for- tion of singlet oxygen [16–18].

Singlet O reacts efficiently with organic dyes, 2 amino acids, and nucleobases [19, 20].

Oxidized dyes are no longer fluor- cent; oxidative damage impairs the folding and function of biomolecules.

In the presence of saturating dissolved O , blinking of fluorescent dyes is sup- 2 pressed, but oxidative damage to dyes and biomolecules is rapid.

Enzymatic O -scavenging systems are commonly employed to ameliorate dye instability. 2 Small molecules are often employed to suppress blinking at low O levels.