Antisense oligonucleotides and ribozymes as gene knockdown reagents

Synthetic single-stranded oligodeoxynucleotides can act as gene-specific antisense reagents, which target in a sequence-specific manner to mRNA through base-pairing to inhibit the translation of a specific protein. Ribozymes, or catalytic RNA, are chimeric RNA molecules in which two stretches of antisense RNA, sharing complementarity with the RNA sequence to be cleaved, flank a nucleolytic motif. Hybridization by base pairing should in theory provide high specificity and affinity. Both oligonucleotides and ribozymes also have particular usefulness as gene therapy agents. Optimal design of antisense oligonucleotides is critical and requires good knowledge of the target to be addressed. Antisense oligonucleotides have to meet criteria of length, type of modification and mode of action, which in turn determine the affinity and specificity of the reagent. In order to bind specifically to a given RNA, 12-50mers are the most often used. Since natural oligonucleotides are susceptible to exonuclease and endonucleases, various chemical modifications are used to protect synthetic oligonucleotides from degradation, such as exchange of phosphodiester internucleotide linkages by phosphorothioate or polyamides (as in peptide nucleic acids). Target selection plays an important role in finding the optimal binding site. The structure of RNA is of great importance for its accessibility. It has been shown that antisense oligonucleotides bind well to only a limited number of sites. RNA molecules fold into complex secondary and tertiary structures; site selection could in principle be based on secondary structure predictions, but since our knowledge of folding and stability of RNA is rather limited, experimental approaches based either on hybridization techniques or on RNase H activity are advisable.

The major aims of collaborative European antisense research within this programme are to address issues of the design of antisense oligonucleotides, particularly RNA target accessibility and site selection, to create standardized antisense/ribozyme application protocols and to develop a database of successful constructs and assays. These aims clearly link chemistry, cell biology and bioinformatics.