The extinction coefficient of DNA and RNA refers to the ability of these molecules to absorb ultraviolet (UV) light at a specific wavelength. The extinction coefficient allows measuring the concentration of nucleic acids in a sample, as the amount of UV absorption is directly proportional to the concentration of nucleic acid molecules in the sample.
The extinction coefficient of DNA and RNA depends on the nucleotide composition and the wavelength of the UV light used for measurement. The extinction coefficient is generally expressed in absorbance units per unit concentration, typically in liters per mole per centimeter (L/mol/cm). The most used wavelength for measuring the extinction coefficient of nucleic acids is 260 nm.
Also, specific functional groups in the nucleotide bases, such as the amino and keto groups, influence the extinction coefficient of DNA and RNA. In general, purine bases (adenine and guanine) absorb more UV light than pyrimidine bases (cytosine, thymine, and uracil) due to an additional double bond in their ring structure. As a result, DNA and RNA molecules containing more purine bases have higher extinction coefficients than those containing more pyrimidine bases.
The extinction coefficient of DNA is typically higher than that of RNA due to the presence of an additional hydroxyl group in RNA's ribose sugar. This hydroxyl group can interfere with UV absorption and reduce the extinction coefficient of RNA compared to DNA.
In addition to measuring the concentration of nucleic acids in a sample, the extinction coefficient also allows the determination of the purity of a nucleic acid preparation. Pure DNA or RNA will have a high extinction coefficient at 260 nm and a low extinction coefficient at 280 nm, while impurities such as proteins will absorb more UV light at 280 nm.
The extinction coefficient of DNA and RNA is a valuable parameter for measuring the concentration and purity of nucleic acid samples. The nucleotide composition and the wavelength of the UV light used for measurement influence the calculated extinction coefficient. The extinction coefficient is typically higher for DNA than for RNA due to differences in their chemical structure.
Nucleic acids, both DNA and RNA, contain conjugated double bonds in their purine and pyrimidine rings with a specific absorption peak at 260 nm. According to the Beer-Lambert law, the amount of energy absorbed to a particular wavelength is a function of the concentration of the absorbing material.
The extinction coefficient of double-stranded DNA is less than the sum of the extinction coefficients of the individual strands. This property is known as hypochromicity caused by base stacking in dsDNA.
The Beer-Lambert law is expressed as
I = I010-ɛdc,
Where I is the intensity of transmitted light; I0 is the intensity of the incident light; ɛ is the molecular extinction coefficient (also known as the molecular absorption coefficient); d is the optical path length (in cm); c is the concentration of the absorbing material (in moles/liter); and ɛ is numerically equal to the absorbance of a 1 M solution in 1-cm light path expressed as M-1cm-1.
Absorbance data collected are generally reported as absorbance [log(I/I0)]. Where d = 1 cm, A is called the optical density or OD at a particular wavelength:
ODl = ɛc
The Beer-Lambert law is valid for at least up to an OD = 2. The molecular extinction coefficient (ɛ) for nucleic acids decreases as adjacent purines and pyrimidines' ring system becomes stacked in a polynucleotide chain. The value for ɛ falls in the following order:
Free base > small oligonucleotides > single-stranded nucleic acids > double-stranded nucleic acids.
Absorbance measurements at 260 nm permit the direct calculation of nucleic acid concentration in a sample:
RNA: μg/ml = A260 × dilution × 40.0
Where A260 = absorbance (in optical densities) at 260 nm, dilution = dilution factor (usually 200–500), 40.0 = average extinction coefficient of RNA.
A similar approach allows for determining the concentration of a DNA sample:
DNA: μg/ml = A260 × dilution × 50.0
Where A260 = absorbance (in optical densities) at 260 nm, dilution = dilution factor (usually 200–500), 50.0 = average extinction coefficient of double-stranded DNA.
It is important to note that concentrations calculated from UV 260 nm absorbance are only accurate for purified DNA and RNA molecules. The solution's ionic strength and pH affect the extinction coefficients of nucleic acids. Controlling the pH of the solution helps to achieve accurate results. Also, the ionic strength of the solution needs to be low (<0.1 M).
dsDNA: The molar extinction coefficient of double-stranded DNA at 260 nm is 6.6.
ssDNA and ssRNA: The molar extinction coefficient of single-stranded DNA and RNA is ~7.4.
dsDNA: For double-stranded DNA, the average coefficient is 50 (mg/mL)-1cm-1.
ssDNA or ssRNA: The average coefficient is 38 (mg/mL)-1cm-1.
ddDNA: 1 OD260 unit = 50 mg/ml.
ssDNA and ssRNA: 1 OD260 unit = 38 mg/ml.
Cavaluzzi and Borer, in 2004, noticed that nearly all of the previously published extinction coefficients for the nucleoside-5′-monophosphates are too large, with an error of as much as 7%. The researchers noted that the accuracy of the results is potentially limited by uncertainties in the material's extinction coefficient, ε, in the Beer–Lambert law: A = ε·C·l, where l is the pathlength of the cuvette (ε = A/C·l).
Due to turbidity, these uncertainties may arise from UV-absorbing impurities, pH effects, and light scattering. Other possible causes for deviations from a linear Beer's law behavior are reorientation of the chromophores due to base pairing, stacking, and other conformational changes such as aggregation and formation of complexes with ligands.
A recent study by Nwoekeoji et al. determined the extinction coefficient (ɛ) for dsRNA to be between 46.18 and 47.29 μg mL−1/A260 by measuring the change in absorbance of samples upon thermal denaturation in the presence of DMSO. The research group determined the hypochromicity of the oligonucleotide or complex nucleic acid structure to allow for accurate quantification. Using the chemical denaturant dimethyl sulfoxide and a short thermal denaturation step prevented the renaturation of the duplex nucleic acids (dsDNA/RNA).
More recently, Strezsak et al. developed a nucleic acid digestion method to digest double- and single-stranded RNA and DNA into nucleosides. A reversed-phase HPLC/UV method allowed the separation and quantitation of the monomeric nucleosides.
This method allowed the researchers to calculate the absorptivity coefficient (a proxy for the extinction coefficient) for dsRNA to be 45.9 ± 0.52 μg mL-1/A260.
However, the scientists noticed that the sequence design could dramatically change the extinction coefficient of the molecule. A 5% reduction in the calculated extinction coefficient was observed for molecules with ssRNA overhangs.
Cavaluzzi MJ, Borer PN. Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 2004 Jan 13;32(1):e13. [PMC]
Green & Sambrook; Molecular Cloning. A Laboratory Manual. 4th Edition. Page 78 to 79. Cold Spring Harbor, NY. (www.molecularcloning.org)
Nwokeoji AO, Kilby PM, Portwood DE, Dickman MJ. Accurate Quantification of Nucleic Acids Using Hypochromicity Measurements in Conjunction with UV Spectrophotometry. Anal Chem. 2017 Dec 19;89(24):13567-13574. [ACS]
Shen, Chang-Hui; Detection and Analysis of Nucleic Acids. 2019 in Diagnostic Molecular Biology (Book).[sciencedirect.com]
Strezsak SR, Beuning PJ, Skizim NJ. Complete enzymatic digestion of double-stranded RNA to nucleosides enables accurate quantification of dsRNA. Anal Methods. 2021 Jan 21;13(2):179-185. [RSC]