Niels Langkjær, University of Southern Denmark, Denmark
Bolle Ravindra Babu, University of Southern Denmark, Denmark
Torben Højland, University of Southern Denmark, Denmark
Mikhail Abramov, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
Arthur Van Aerschot, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
Dalibor Odadzic, Institute of Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University, Germany
Romualdas Smicius, Institute of Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University, Germany
Jens Haas, Institute of Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University, Germany
Cordula Andree, Max Planck Institute of Molecular Cell Biology and Genetics, Germany
Jharna Barman, Uppsala University, Sweden
Malgorzata Wenska, Uppsala University, Sweden
Puneet Srivastava, Uppsala University, Sweden
Chuanzheng Zhou, Uppsala University, Sweden
Dmytro Honcharenko, Uppsala University, Sweden
Simone Hess, Department of Molecular Biology, Max Planck Institute for Infection Biology, Germany
Elke Müller, Department of Molecular Biology, Max Planck Institute for Infection Biology, Germany
Georgii V Bobkov, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Russian Federation
Sergey N Mikhailov, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Russian Federation
Eugenio Fava, Max Planck Institute of Molecular Cell Biology and Genetics, Germany
Thomas F Meyer, Department of Molecular Biology, Max Planck Institute for Infection Biology, BerlinDepartment of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany
Jyoti Chattopadhyaya, Uppsala University, Sweden
Marino Zerial, Max Planck Institute of Molecular Cell Biology and Genetics, Germany
Joachim W Engels, Institute of Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University, Germany
Piet Herdewijn, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium
Jesper Wengel, University of Southern Denmark, Denmark
The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.