GSK-503 However, these approaches are not readily applicable to shRNA design. Substitutions for optimization of duplex asymmetry can change shRNAs enzymatic cleavage patterns, while nucleotide chemical modifications can��t be performed in vivo. The duplex stability threshold for the fully paired antisense strands of efficient siRNAs was discussed recently. It was found that exclusion of siRNAs that form stable duplexes Vigabatrin improves the accuracy of different siRNA prediction models. Thermodynamic evaluation of duplex stability using nearest neighbor parameters is more accurate than those based on evaluation of GC content. We found that siRNA duplexes with identical GC content have different DG values with variations as high as 4 kcal/mol. Why use thermodynamic parameter thresholds instead of weights in predictive models? The disadvantage of regression analysis and similar approaches for predicting molecular efficiency are weight values assigned to each input variable to generate the predictor of siRNA efficiency in the output. These weight values can change as a reflection of concentration changes of siRNA or mRNA components in the cleavage reaction. As a result, weights that are found to be optimal based on the analysis of one experimental database might be far from optimal for another database. Results of regression analysis or similar approaches could be difficult to extrapolate on shRNA design, especially for viral transduction of shRNA constructs. In this case, the cellular concentration of the expected siRNA�Clike cleavage products is most likely smaller than with plasmid or oligonucleotide transfections. Threshold parameters might be adjusted to be less dependent on concentration changes and can be more universal in predicting molecular behavior under different experimental settings. Why does duplex stability of the fully paired siRNA antisense strand affect silencing efficiency? This parameter should not be too high or too low for efficient siRNA functioning. Low duplex stability results in slow formation and short life time of antisense strand-target duplexes, which could be insufficient for RNA cleavage to occur.