Characterization of photoactivated singlet oxygen damage in single-molecule optical trap experiments

Abstract

Optical traps or "tweezers" use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments-the most common biological application of optical tweezers-and may guide the development of more robust experimental protocols.

Figure 1

Dependence of tether lifetime on trap power. (A) Schematic representation of a dsDNA tether with 5′-digoxigenin (labeled DIG) and 5′-biotin (BT) modifications held between an anti-digoxigenin (AD, red) and a streptavidin (SA, blue) microsphere. (B) Tether lifetime versus laser power measured at the AD microsphere. Average lifetimes of tethers formed between 0.86-μm AD and 0.79-μm SA microspheres in traps of identical power (black squares, N = 18–55), and asymmetric power, with the AD microsphere in the low-power trap (open red diamond, N = 23) and high-power trap (open dark yellow circle, N = 22). Open symbols represent tethers under identical total trap power. Average lifetime of tethers formed between 2.1-μm AD and SA microspheres (cyan circle, N = 29), between a 2.1-μm AD microsphere and a 0.79-μm SA microsphere (blue X, N = 16) and between two 0.79-μm SA microspheres through dual biotin-streptavidin linkages (green diamond, N = 8). Inset: Above data plotted as a function of laser power measured at the SA microsphere. All tethers were held at tensions of 10–20 pN (14 ± 3 pN; mean ± SD, N = 268). Error bars = SE. Power-law fit to tether lifetime versus trap power measured at the AD microsphere yields the equation t = A × P^B with A = (3.9 ± 2.0) × 10⁵ s/mW and B = −1.66 ± 0.12, (χ² = 13.7).

Figure 2

Dependence of tether forming efficiencies on trap irradiation. Tether formation was attempted in microsphere pairs in two configurations: 0.79-μm DNA-coated SA with 0.86-μm bare AD microspheres (blue bars; ∼20 mol/microsphere), or DNA-coated AD with bare SA microspheres (red bars; ∼30 mol/microsphere). Tethering efficiencies were measured for DNA-coated SA (blue bars 1–3), AD (blue bars 4–6), SA (red bars 1–3), and DNA-coated AD (red bars 4–6) microspheres under the following conditions: after initial trapping at low, 100 mW laser power (denoted 0 LO), after 10 min of exposure to high, 350 mW power (10 HI), or after 10 min of exposure to low, 100 mW power (10 LO). Each trial involved a tethering attempt with a new, unexposed complementary microsphere. Tethering efficiencies were calculated using the Laplace best estimator (50) (S + 1)/(N + 2), where S is the number of successes and N is the number of trials. This estimator is considered better than the maximum-likelihood S/N, when N is small. Error bars = 95% confidence intervals from the adjusted Wald method (51).

Figure 3

Low frequency noise as a function of DNA on microspheres. (A) Power spectra for a SA microsphere coated with 0 (black), ∼30 (red), ∼80 (green), and ∼400 molecules (blue) of DNA exposed to 300 mW laser power. (B) Excess integrated noise between 0 and 100 Hz as a function of the number DNA molecules on the microsphere. Each data point represents the average from nine power spectra from three separate microspheres. Error bars = SE.

Figure 4

DNA dissociation from microspheres. (A) Power spectra of a heavily DNA-coated AD microsphere (∼230 mol/ microsphere) at t = 4 min (blue), 14 min (green), and 34 min (red) and an AD microsphere with no DNA (black). (B) Excess integrated noise between 0 and 100 Hz as a function of time for 0.79-μm SA (blue squares), 0.86-μm AD (red circles), and 0.97-μm SA silica (black diamonds) DNA-coated microspheres exposed to 350 mW of laser power. Error bars = SE from five power spectra. Red, blue, and black lines are trend lines to guide the eye.

Figure 5

SOSG fluorescence in optically trapped microspheres. (Top) Brightfield images. (Center) Fluorescence images at 535 nm. (Bottom) Fluorescence image line scans. (A) 0.79-μm SA polystyrene microsphere without SOSG. (B) No microsphere with SOSG. (C) 0.79-μm SA polystyrene microsphere with SOSG. (D) 2.1-μm SA polystyrene microsphere with SOSG. (E) 0.78-μm silica microsphere with SOSG. Scale bar = 1 μm. A trap power of 390 mW was used in all the images.

Figure 6

Oxidative damage to DNA hairpin. (A) Hairpin force-extension behavior under aerobic conditions (without GODCAT): stretching curves (black), relaxation curves after holding the hairpin folded at t = 0 s (red), 75 s (green), 200 s (blue), and 220 s (cyan). (B) Hairpin force-extension behavior under anaerobic conditions (with GODCAT): stretching curves (black), and relaxation curves at t = 0 s (red), 150 s (green), and 330 s (blue). All force-extension curves obtained at a pulling rate of 2 pN/s. (C) Increase in hysteresis area as a function of time for a DNA hairpin stretched under anaerobic condition (red squares), and aerobic conditions (green diamonds). Error bars = SE from 49 force-extension curves of two tethers. Red line and green curve are trend lines to guide the eye.