The colorimetric determination of selectively cleaved adenosines and guanosines in DNA oligomers using bicinchoninic acid and copper.
Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this proof-of-principle study, we demonstrate that bicinchoninic acid (BCA) and copper, when combined with purine-specific chemical cleavage reactions, can be a colorimetric probe for the identification and quantification of adenosines and/or guanosines in single-stranded DNA oligomers, even in the presence of pyrimidines. Furthermore, the reactions are stoichiometric, which allows for the quantification of the number of adenosines and/or guanosines in these oligomers. Because the BCA/copper reagent detects the reducing sugar, 2-deoxyribose, that results from the chemical cleavage of a given nucleotide's N-glycosidic bond, these colorimetric assays are effectively detecting apurinic sites in DNA oligomers, which are known to occur via DNA damage in biological systems. We demonstrate that simple digital analysis of the color-changing chromophore (BCA/copper) is all that is necessary to obtain quantifiable and reproducible data, which indicates that these assays should be broadly accessible.
Department of Chemistry, University of Kentucky, 550 Rose Street, Lexington, KY, 40506, USA.
Elizabeth M
EM
Thomas
Department of Chemistry, University of Kentucky, 550 Rose Street, Lexington, KY, 40506, USA. testa@uky.edu.
Stephen M
SM
Testa
D000241:Adenosine / Q000737:chemistry*; D003116:Color; D003124:Colorimetry; D003300:Copper / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D006151:Guanosine / Q000737:chemistry*; D011804:Quinolines / Q000737:chemistry*
D004277:DNA, Single-Stranded; D011804:Quinolines; D006151:Guanosine; D003300:Copper; C047117:bicinchoninic acid; D000241:Adenosine
Apurinic; BCA assay; Colorimetry; Copper; DNA