Method for determining immunostimulatory activity of RNA oligonucleotides

A method for determining the immunostimulatory activity of an RNA oligonucleotide, comprising the steps of:

• (a) complexing the RNA oligonucleotide with a complexation agent;
• (b) contacting a cell with the complexed RNA oligonucleotide, wherein the cell expresses TLR7, TLR8, or both TLR7 and TLR8;
• and
• (c) determining the amount of IFN-α produced by the cell of step (b), an increase of IFN-α production indicating immunostimulatory activity of the RNA oligonucleotide, wherein the RNA oligonucleotide has an IFN-α score of at least 1.4909×n+31.014, where n is length of the RNA oligonucleotide and IFN-α score is calculated by the method comprising the steps of:
• (a) identifying all possible 3-nucleotide (3mer) motifs contained in the oligonucleotide;
• (b) assigning an IFN-α point score for each individual 3mer motif:
• (i) for a 3mer motif which appears in Table 7, assigning an IFN-α point score according to Table 7;
• (ii) for a 3mer motif which does not appear in Table 7, assigning an IFN-α point score of 0;
• and
• (c) assigning the sum of the IFN-α point scores of individual 3mer motifs as the IFN-α score of the oligonucleotide.