Widespread nuclease contamination in commonly used oxygen-scavenging systems

To the Editor

Much to our surprise, we observed substantial DNA nuclease activity with both the glucose-coupled glucose oxidase/catalase (GODCAT)^1,2 and the protocatechuic acid (PCA)–protocatechuate-3,4-dioxygenase (PCD)³ oxygen-scavenging systems (OSSs) that are widely used for single-molecule (SM) studies (Fig. 1a and Supplementary Figs. 1–3). The GODCAT system also displayed non-specific DNA binding/ aggregation activity, a fluorescent cofactor and single-stranded and/or double-stranded DNA endo- and exonuclease activities (Supplementary Figs. 2–4 and Supplementary Notes 1–4). In addition, nearly all the OSS enzymes from multiple commercial sources (Supplementary Table 1, Supplementary Methods) displayed residual Mg²⁺-dependent nuclease contaminants (Supplementary Fig. 5a–c). The commonly used CAT^a,b and PCD^a enzymes contained the most impurities and the highest level of nuclease activity, whereas CAT^c and PCD^b seemed to be the least contaminated commercially available OSS components.

Figure 1.

Nuclease contamination of oxygen-scavenging enzymes; purification and analysis of PCD. (a) Analysis of endonuclease activity in different OSS components. The resulting products include the following: SC, supercoiled monomer; NC, nicked circular monomer; ND, nicked dimer. (b) Normalized absorbance at 280 nm (light blue shaded area), percent PCD activity (blue) and percent endonuclease activity (red) of Superose 6 gel filtration fractions. (c) Kinetics of Cy3 photobleaching at different purified PCD^a concentrations. (d) The calculated half-life of Cy3 with the purified PCD^a OSS in the imaging buffer. The half-life of Cy3 corresponds to a 50% survival probability in c. Error bars denote ±s.e. (e) Extended analysis of the survival probability of Cy3 in the presence of 10 nM purified PCD^a OSS (see main text).

These observations suggested that DNA binding and/or aggregation, nuclease and other contaminants should be removed from the OSS before use in DNA-SM analysis. We developed a straightforward Superose 6 size-exclusion chromatography purification method for PCD^a that takes advantage of the enzyme’s large reported size, with 12 α-dimer (23.3 kDa) and β-dimer kDa) subunits⁴ (Supplementary Notes 5 and 6). The majority of nuclease activity eluted as an ~40-kDa protein, with a slightly higher molecular weight shoulder at ~100 kDa (Fig. 1b and Supplementary Fig. 6), whereas peak PCD enzyme activity corresponded with the αβ-subunits, eluting at 417 kDa (Fig. 1b and Supplementary Figs. 6 and 7). The pooled PCD fractions (43–51) were free of nuclease activity and did not inhibit several DNA metabolic reactions even at high concentrations (100 nM; Supplementary Figs. 5 and 8–10 and Supplementary Note 7).

Using SM photobleaching analysis, we counted and analyzed the number of Cy3 spots on a 3,429-μm² image area as a function of irradiation time at different PCD concentrations (Fig. 1c–e and Supplementary Fig. 11). We observed a PCD concentration–dependent increase in Cy3 half-life, in which Cy3 fluorescence loss at 10 nM PCD was negligible after 4 min of irradiation (Fig. 1c and Supplementary Fig. 11). An extended analysis suggested an apparent photobleaching rate constant of 10 nM PCD (k_pb = 0.016 ± 0.003 min−1; s.e. from the fitting) that resulted in an estimated half-life of Cy3 of ~2,600 s (43 min) (Fig. 1e), which substantially exceeds observations from most SM kinetic studies⁵. Moreover, we observed very little ‘blinking’ of Cy3, similar to previous reports³, suggesting a remarkable additional stability that is capable of enhancing SM analysis (data not shown).

Finally, GOD^aCAT^a and PCD^a are the most common OSS enzymes used in SM DNA and RNA analysis. Our surveys strongly suggest that these enzymes could influence SM observations as a result of multiple contaminants. We recommend the use of commercial PCD^b or purified PCD^a, as outlined here for DNA- or RNA-based SM studies (Fig. 1 and Supplementary Fig. 5). Although the commercial CAT^c preparation is relatively free of nuclease, there does not seem to be a commercial GOD product or method of purification that will provide a nuclease-free OSS with these enzymes.