How can I calculate the Tm increase induced by ZNA®s?
The relative Tm increase induced by ZNA or other duplex stabilizing groups is naturally larger on shorter oligonucleotides used as primers or probes. Hence, we offer ZNA primers as short as 10mers and dual-labelled probes as short as 8mers!
The Tm increases significantly and quite linearly with the number of grafted ZNA® spermine units. The approximate Tm of a ZNA® can be calculated applying the following equation:
Tm (ZNA®) = Tm (DNA) + 36z/(N-3.2) (1)
z: number of cationic units
N: number of nucleotides
Sequence 5´ATATATAT 3´ 8mer Tm(DNA) = 16 °C
ZNA-2 building block
Tm(ZNA) = 16 + 36*2/(8-3.2) = 31 °C
ZNA-2 modification almost doubles Tm; Tm increase of approx 15°C!
Paying respect to the global charge of the ZNA-oligonucleotide-oligocation conjugates raising solubility issues, we additionally offer ZNA-2 and ZNA-3 (cationic) building blocks for (anionic) primers ranging from 8 to 15mers, and dual labeled probes ranging from 10 to 17mers, respectively. Attachment of ZNA-4 and ZNA-5 building blocks to primers is allowed from 16mers (ZNA-4), and 20mers (ZNA-5), respectively. Attachment of ZNA-4 and ZNA-5 building blocks to dual labeled probes is allowed from 18mers (ZNA-4), and 22mers (ZNA-5), respectively.
Considering the ZNA solubility issues and the global charge of a ZNA-oligonucleotide conjugate, a ratio of 1 spermine each 4 nucleotides is appropriate. Accordingly, we offer ZNA-4 building blocks for an unmodified 16mer, for example. In case of dual labeled probes, ZNA-4 is most suitable for a 18mer.
The Tm can be additionally increased, while maintaining specificity, by incorporation of our base analogues:
- C-5 propynyl-dC (pdC) raising melting temperature by ~2.8 °C per substitution
- C-5 propynyl-dU (pdU) raising melting temperature by ~1.7 °C per substitution.
There are many different algorithms to calculate the Tm of an oligo. All are just approximations of the actual Tm of a specific oligo under specific conditions (salt concentrations, pH, temperature, sequence composition, oligo length and other biophysical/ biochemical parameters and reaction conditions). Optimization is always recommended.