THEORETICAL AND EXPERIMENTAL APPROACHES TO DESIGN EFFECTIVE ANTISENSE OLIGONUCLEOTIDES

Georg Sczakiel

Forschungsschwerpunkt Angewandte Tumorvirologie - F0200, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Materials and methods

3.1. Computer algorithms
3.2. Generation of limited antisense sequence space
3.3. Selection of fast annealing antisense species

4. Results and discussion

4.1. Target rna structures are important for efficacy of antisense oligonucleotides
4.2. Theoretical versus experimental approaches: advantages and disadvantages
4.3. Current experimental protocols to design asodn

4.3.1. Protocols in the use of pools of oligonucleotides
4.3.2. Experimental kinetic probing of target structures with pools of rna species

4.3.2.1. Hpv 16-directed asodn: kinetic probing and the design of an effective antisense 20mer
4.3.2.2. Antisense oligonucleotides against bcr-abl sequences: kinetic probing and efficacy in human cells

4.4. Computational approaches to design antisense oligonucleotides

5. Acknowledgement

6. References

1. Abstract

Among the large number of possible antisense oligonucleotides (asODN) against a given target nucleic acid, only a small number of species seems to give rise to satisfactorily strong inhibition of target gene expression in living cells. Therefore much attention is paid to strategies that help to successfully design effective asODN. Here, selected experimental approaches and theoretical concepts will be briefly described that have been developed to increase the probability of success in the use of asODN. Advantages and disadvantages of these strategies will be compared and the relatively new and controversially discussed concept of a theoretical and computer-supported design of effective asODN will be addressed.