ABSTRACT
Antisense oligonucleotides (AON) are being developed for a wide array of therapeutic applications. Significant improvements in their serum stability, target affinity, and safety profile have been achieved with the development of chemically modified oligonucleotides. Here, we compared 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)-containing AONs with phosphorothioate oligodeoxynucleotides (PS-DNA), 2'-O-methyl-RNA/DNA chimeras and short interfering RNAs (siRNA) with respect to their target knockdown efficacy, duration of action and resistance to nuclease degradation. Results show that two different configurations of FANA/DNA chimeras (altimers and gapmers) were found to have potent antisense activity. Specific target inhibition was observed with both FANA configurations with an estimated EC50 value comparable to that of an siRNA but 20-to 100-fold lower than the other commonly used AONs. Moreover, the FANA/DNA chimeras showed increased serum stability that was correlated with sustained antisense activity for up to 4 days. Taken together, these results indicate that chimeric FANA/DNA AONs are promising new tools for therapeutic gene silencing when increased potency and duration of action are required.
Subject(s)
Arabinonucleotides/pharmacology , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacology , Drug Stability , Gene Silencing , HeLa Cells , Humans , Inhibitory Concentration 50 , Luciferases/antagonists & inhibitors , Luciferases/genetics , RNA, Small Interfering/pharmacology , Structure-Activity Relationship , Thionucleotides/pharmacology , Time FactorsABSTRACT
Ribonucleases H are complex enzymes whose functions are not clearly understood, further compounded by the fact that multiple forms of the enzyme are present in various organisms. They are known to recognize and degrade the ribonucleic acid (RNA) strand of numerous deoxyribonucleic acid (DNA)-RNA duplex substrates, and so may provide a unique mode of therapeutic intervention at the genetic level of virtually any disease. We have therefore set out detailed procedures for conducting routine assays with almost any one of this family of enzymes by a straightforward assay aimed at identifying novel enzyme-activating antisense oligonucleotides (AONs). The procedures described herein should enable easy identification of potent AON molecules, provided that the RNA is appropriately labeled for subsequent visualization following the guidelines set forth in this protocol.
Subject(s)
Oligonucleotides, Antisense/metabolism , RNA/metabolism , Ribonuclease H/metabolism , Base Sequence , DNA Primers , Oligonucleotides, Antisense/chemistry , RNA/chemistryABSTRACT
We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2''-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2''-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.
Subject(s)
Oligodeoxyribonucleotides/chemical synthesis , Oligoribonucleotides/chemical synthesis , Circular Dichroism , DNA/chemistry , Fluorine , Nucleic Acid Conformation , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/chemistry , Oligoribonucleotides/chemistry , RNA/chemistryABSTRACT
A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and non-natural (i.e., arabinose, xylose and acyclic nucleoside) branchpoints in order to examine the effect of sugar conformation and phosphodiester configuration on yDBR hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a cis-arrangement of phosphate groups at the branchpoint (i.e., ribose) thereby discriminating between all other configurations.
Subject(s)
RNA Splicing/genetics , RNA, Fungal/chemistry , RNA, Fungal/genetics , Nucleic Acid Conformation , RNA Nucleotidyltransferases/metabolism , Saccharomyces cerevisiae/enzymologyABSTRACT
The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.
Subject(s)
Arabinose/analogs & derivatives , DNA, Antisense/chemistry , Oligonucleotides, Antisense/chemistry , RNA, Complementary/chemistry , Ribonuclease H/chemistry , Arabinose/chemistry , DNA/chemistry , DNA/metabolism , DNA, Antisense/chemical synthesis , Escherichia coli/enzymology , Genes, ras , Humans , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/chemistry , Oligonucleotides, Antisense/chemical synthesis , RNA, Complementary/metabolism , Ribonuclease H/metabolism , Structure-Activity RelationshipABSTRACT
A comparison of carbohydrate modified nucleic acids has identified key structural characteristics in antisense oligonucleotides (AON) that are necessary for sufficient clinical utility, including increased duplex stability towards RNA complements and improved hydrolytic resistance towards general serum and cellular nucleases. As such, the exogenous addition of short, synthetic oligonucleotides can influence cellular RNA metabolism at any or all levels of replication, transcription or translation by tight and specific hybridization with a chosen target and subsequently stop further function at that site. Furthermore, appropriate modification of the sugar residue may prove to be a vital design element in future AONs that operate by promoting enzyme assisted catalytic destruction of the mRNA target. Unfortunately, many of the current AON designs have provided little insight on the particular structural role of the AON towards enzymatic discrimination of the resultant hybrid. The use of RNase H as a cellular vehicle to assist the inhibitory potency of an AON as well as possible ways of enhancing activity in pre-existing antisense candidates are presented. Of the emerging criteria in this aspect, a balance between flexibility and rigidity within the AON appears to be a critical mediator of the RNase H assisted antisense effect. Accordingly, this review describes the conformational features and selected biological attributes of some of the more prominent AON contenders with a focus on the conformational criteria by which ribonuclease H activity is recruited to a particular hybrid target.
Subject(s)
Oligonucleotides, Antisense/chemistry , Pentoses/chemistry , Humans , Nucleic Acid Conformation , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , RNA, Messenger/antagonists & inhibitors , Ribonuclease H/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
This unit describes the chemical synthesis of 2'-deoxy-2'-fluoro-b-D-oligoarabinonucleotides (2'F-ANA), both with phosphodiester and phosphorothioate linkages. The protocols described herein include araF phosphoramidite preparation, assembly on DNA synthesizers, and final deprotection and purification of oligonucleotides.
