Spectrophotometric analysis of nucleic acids: oxygenation-dependant hyperchromism of DNA

Abstract

The absorbance at 260 nm (A(260)) is ubiquitously used for nucleic acid quantification. We show that following oxygenation, DNA solutions experience alterations in both spectral properties (hyperchromism in the UV region, lambda(max) 260 nm) and DNA conformation. The spectral changes caused by oxygen-DNA complexation are stable for at least several weeks at room temperature or several hours at 37 degrees C, but are also reversible by purging with nitrogen. Our data indicate that DNA in working solutions might already exist in the oxygen-complexed state, potentially confounding spectrophotometric analyses. Further, the presence of these complexes does not appear to impart cell toxicity in vitro or affect the biophysical functional behaviour (e.g. hybridisation) of DNA. Interestingly, our work also suggests that hybridisation could determine a release of bound oxygen, a phenomenon that could open the way to the use of such systems as oxygen carriers.

Nucleic Acids in Atypical Conditions

“…To gases saturation, DNA solutions (20 °С) were bubbled with gases; gases were supplied at 1,05 atmospheric pressure, for half an hour, in [2,3].…”

“…The content of these gases in DNA working solutions is very variable; 3. The oxygenation of DNA solutions accompany to their UV hyperchromism, in [2,3]. Separately need to say about hydrogen, because its donors (glucose, fats, etc.)…”

An Alternative Strategy in Cancer Chemotherapy, Aimed Not at Killing Cancer Cells, but the Recovery of Their DNA, Modified by Active Oxygen

“…That oxidized DNA still loses atomic oxygen in the presence of chloride anions can be seen by using spectral methods, because a characteristic feature of the oxidized DNA is the peak with a maximum at 260 nm, which is detected in the UV absorption spectra of aqueous solutions of DNA [7][8][9][10][11]. It is known that this peak increases rapidly when modification of DNA by active oxygen and also decreases rapidly in the process of degassing or deoxygenating aqueous solutions pre-oxidized DNA [9][10][11].…”

“…It is also important that oxygen is very easily detaches from the DNA: for example, this occurs when degassing of aqueous solutions of DNA or sparging with nitrogen [8][9][10][11]. Thus, the relationship between oxygen and DNA damaged by reactive oxygen (for the sake of brevity, we will call it oxidized DNA), is weak.…”

UV Absorbance of Aqueous DNA

“…Furthermore, since water obtains the negative charge (potential) during evaporation, in [5][6][7][8], can to conclude that state and UV absorbance of aqueous DNA should be depend on the conditions, in which the DNA working solutions are stored or water used for their preparation. Thus, an A 260 of DNA solution depends not only on content of oxygen, in [9][10][11]. In this connection, it should be recognized: A 260 cannot be used to accurately determine the concentration of DNA.…”