Richard Owczarzy
Extinction (absorption) coefficients of nucleic acids calculated at 260 nm
We have developed the method1 that predicts oligonucleotide UV spectrum as well as extinction coefficients for both single-stranded and double-stranded DNAs. The most accurate approach is based on the nearest-neighbor model and its published parameters2,3 for DNA and RNA. These parameters are presented in the table below and were determined at wavelength of 260 nm, and neutral pH. The average error of calculated extinction coefficients was shown4,5,6 to be around 4 % under these conditions. For example, the extinction coefficient (ε) of linear oligonucleotide 5'-dATGCTTC-3' is,
If any modifying chemical group is attached to an oligonucleotide, extinction coefficient of the group needs to be added.
The table of extinction coefficients [liter/(mol.cm)] for DNA and RNA oligonucleotides.
| DNA | RNA | ||
|---|---|---|---|
| Stack or monomer | Extinction coefficient | Stack or monomer | Extinction coefficient |
| pdA | 15400 | pA | 15400 |
| pdC | 7400 | pC | 7200 |
| pdG | 11500 | pG | 11500 |
| pdT | 8700 | pU | 9900 |
| dApdA | 27400 | ApA | 27400 |
| dApdC | 21200 | AdC | 21000 |
| dApdG | 25000 | ApG | 25000 |
| dApdT | 22800 | ApU | 24000 |
| dCpdA | 21200 | CpA | 21000 |
| dCpdC | 14600 | CpC | 14200 |
| dCpdG | 18000 | CpG | 17800 |
| dCpdT | 15200 | CpU | 16200 |
| dGpdA | 25200 | GpA | 25200 |
| dGpdC | 17600 | GpC | 17400 |
| dGpdG | 21600 | GpG | 21600 |
| dGpdT | 20000 | GpU | 21200 |
| dTpdA | 23400 | UpA | 24600 |
| dTpdC | 16200 | UpC | 17200 |
| dTpdG | 19000 | UpG | 20000 |
| dTpdT | 16800 | UpU | 19600 |
Extinction coefficients are typically used to determine oligonucleotide concentrations from the Lambert-Beer law,
Absorbance of single stranded oligonucleotide is typically measured in a buffer of neutral pH and low salt concentrations (less than 10mM). High concentrations of cations may promote oligonucleotide folding. This is even more likely for RNA oligonucleotides, therefore, it is recommended to disrupt folding by heating up solution to temperature of 85oC for absorbance measurements7.
The extinction coefficient of any duplex DNA (εD) is less than the sum of the extinction coefficients of its complementary strands (εS1, εS2). This hypochromicity effect1 can be taken into account,
where fAT and fGC are fractions of AT and GC base pairs, respectively.
- Tataurov A.V., You Y., and Owczarzy R. (2008) Predicting ultraviolet spectrum of single stranded and double stranded deoxyribonucleic acids, Biophys. Chem. 133, 66-70.
- Cantor C.R., Warshaw M.M., and Shapiro H. (1970) Oligonucleotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleotides, Biopolymers 9, 1059-1077.
- Fasman, G.D. (Ed.) (1975) Handbook of Biochemistry and Molecular Biology, Volume 1: Nucleic Acids, pp 589, 3rd edition, CRC Press.
- Kallansrud G., and Ward B. (1996) A comparison of measured and calculated single- and double-stranded oligodeoxynucleotide extinction coefficients, Anal. Biochem. 236, 134-138.
- Murphy J.H, and Trapane T.L. (1996) Concentration and extinction coefficient determination for oligonucleotides and analogs using a general phosphate analysis, Anal. Biochem. 240, 273-282.
- Cavaluzzi M.J., and Borer P.N. (2004) Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA, Nucleic Acids Res. 32, e13.
- Xia T., SantaLucia J., Jr., Burkard M.E., Kierzek R., Schroeder S.J., Jiao X., Cox C., and Turner D.H. (1998) Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs, Biochemistry 37, 14719-14735.