Modified oligonucleotides also need special consideration to ensure accuracy when calculating molecular weight, extinction coefficient (ε260), and melting temperature (TM). Modifications can change oligo mass, and sometimes alter UV absorbance or TM.
Molecular Weight of an oligo containing an NHS Ester modification (such as 5' Texas Red® NHS Ester) is a sum of molecular weights of native oligo, the fluorophore group, and an amino modifier.
Extinction Coefficients (ε260) of modifications, such as fluorophores and base analogs, are usually added to the ε260 of the native oligonucledotide. Calculations for base analogs (e.g., 5-bromo dC) and conjugated bases (e.g., fluorescein dT) are more complex. First, ε260 of an oligo containing the unmodified base is calculated. An adjustment is made later for the contribution of the modification (fluorophore). Unfortunately, ε260 values are not known for all modifications.
Melting Temperature (TM) can be changed when nucleotides are modified or additional chemical groups are added. For example, introduction of phosphorothioated residues decreases TM significantly. In contrast, LNA nucleotides increase TM. Unfortunately, nearest neighbor thermodynamic parameters have not been determined for a majority of these modifications. Therefore, no accurate parameters and physical models exist that would allow us to calculate melting temperatures for many modified oligonucleotides. Internal base modifications (e.g., biotin-dT), could collide and interfere with the duplex structure. Because the quantitative effects of interference are unknon, they are neglected. If thermodynamic parameters are not available, Oligo Analyzer reports TM values for the unmodified sequence. Melting temperature changes casued by modifications may be approximated from the published literature. If needed, a precise TM can be measured experimentally.