**46k**wrote:

siRNA's are 19-21 long RNA duplexes with 2 base 3' overhangs present in the RNA-interference pathway. I would like to calculate the local internal stability of several RNA duplexes in siRNA, as described in Khvorova et al. (2003) based on (delta G) calculations according to Freier et al (1986). I also want to calculate local stability at other temperatures than 37 degrees (not every bug lives at 37 degr. C) to achieve plots similar to Figure 2 in Khvorova et al. How well does the stability calculation transfer?

In Kvorova et al., they used the proprietary OLIGO software for this purpose. Is there an open-source alternative to this procedure (Khvorova et al.):

The Oligo 5.0 Primer Analysis Software (National Biosciences, Inc., Plymouth, MN) was used for calculations of internal stability profiles using the free energy values in Table 1 (−kcal/mol, adjusted to 25°C).

... The internal stability values reflect the stability of pentamer subsequences within the sequence under investigation and were calculated according to the nearest neighbor method (Freier et al., 1986). For the calculation of the average internal stability values of the terminal four bases on the 3′ end of the molecule, the 19 bp targeting sites were extended based on the target mRNA or miRNA precursor sequences.

I guess the calculation should be rather basic, therefore a formula for the nearest neighbor method of Freier should be sufficient.

Sorry if this is a very basic and partially biochemistry/biophysics question. Edit: Seemingly, it is not at all as easy as I thought, even though any primer designer software and RNA-folding software should internally compute these stabilities. I tried for example Pairfold and mfold, but that outputs only minimum free energy (which is 0 for perfect pairing). Another hint: the claculation should be similar as for "3' Max stability" in eprimer3 but with thermodynamic parameters adjusted for RNA.

**20**• written 6.4 years ago by Michael Dondrup ♦

**46k**