Synthesis and Biophysical and Biological Evaluation of 2′-Modified Antisense Oligonucleotides

153

110

We have previously described structure-activity studies on a 17-mer uniform phosphorothioate antisense sequence targeted to human Ha-ras. In an effort to further improve the pharmacological properties of antisense oligonucleotides, structure-activity studies on this 17-mer sequence were expanded to examine both the effects of replacing phosphorothioate backbone linkages with phosphodiester linkages and the effects of incorporating various 2-sugar modifications into phosphorothioate and phosphodiester oligonucleotides on oligonucleotide stability against nucleases in vitro and on antisense activity in cells. Replacement of three or more phosphorothioate linkages with phosphodiester linkages greatly compromised both nuclease resistance and antisense activity, and these effects correlated directly with the number of phosphodiester linkages incorporated into the oligonucleotide. However, substantial nuclease resistance, sufficient for obtaining potent antisense effects in cells, was conferred to phosphodiester oligonucleotides by incorporation of appropriate 2-alkoxy sugar modifications. Nuclease stability and antisense activity imparted by these sugar modifications in phosphodiester backbones correlated with the size of the 2-alkoxy substituent (pentoxy > propoxy > methoxy > deoxy). Furthermore, antisense activity mediated by oligonucleotides that exhibit partial resistance to nucleolytic degradation was dependent on both oligonucleotide concentration and the duration of oligonucleotide treatment.

Section: Methodsmentioning

confidence: 99%

Nuclease Resistance and Antisense Activity of Modified Oligonucleotides Targeted to Ha-

Monia

Johnston

Sasmor

et al. 1996

153

110

“…Purification of oligonucleotides was also as described previously (29,30). Purified oligonucleotides were greater than 90% full-length material as determined by capillary gel electrophoretic analysis.…”

Section: Materials-t4 Polynucleotide Kinase Was Purchased From Promegmentioning

confidence: 99%

“…Oligonucleotide Synthesis-Synthesis of 2Ј-methoxy, 2Ј-fluoro, 2Ј-propoxy, and deoxy chimeric oligonucleotides was performed using an Applied Biosystems 380B automated DNA synthesizer as described previously (29,30). Purification of oligonucleotides was also as described previously (29,30).…”

Section: Materials-t4 Polynucleotide Kinase Was Purchased From Promegmentioning

confidence: 99%

Properties of Cloned and Expressed Human RNase H1

Lima

Crooke

1999

158

140

We have characterized cloned His-tag human RNase H1. The activity of the enzyme exhibited a bell-shaped response to divalent cations and pH. The optimum conditions for catalysis consisted of 1 mM Mg 2؉ and pH 7-8. In the presence of Mg 2؉ , Mn 2؉ was inhibitory. Human RNase H1 shares many enzymatic properties with Escherichia coli RNase H1. The human enzyme cleaves RNA in a DNA-RNA duplex resulting in products with 5-phosphate and 3-hydroxy termini, can cleave overhanging single strand RNA adjacent to a DNA-RNA duplex, and is unable to cleave substrates in which either the RNA or DNA strand has 2 modifications at the cleavage site. Human RNase H1 binds selectively to "A-form"-type duplexes with approximately 10 -20-fold greater affinity than that observed for E. coli RNase H1. The human enzyme displays a greater initial rate of cleavage of a heteroduplex-containing RNA-phosphorothioate DNA than an RNA-DNA duplex. Unlike the E. coli enzyme, human RNase H1 displays a strong positional preference for cleavage, i.e. it cleaves between 8 and 12 nucleotides from the 5-RNA-3-DNA terminus of the duplex. Within the preferred cleavage site, the enzyme displays modest sequence preference with GU being a preferred dinucleotide. The enzyme is inhibited by single-strand phosphorothioate oligonucleotides and displays no evidence of processivity. The minimum RNA-DNA duplex length that supports cleavage is 6 base pairs, and the minimum RNA-DNA "gap size" that supports cleavage is 5 base pairs.

“…These site-specific cleavage rates (V 0/s ) were measured by quantitating the amount of RNase H digestion for each individual cleavage site as a function of time. For example, site-specific cleavage rates were determined for residues G 26 , G 27 , A 29 , and G 31 of the 22-13 pseudo-half-knot substrate (Fig. 2, A and B).…”

mentioning

confidence: 99%

The Influence of Antisense Oligonucleotide-induced RNA Structure on Escherichia coli RNase H1 Activity

Lima

Mohan

Crooke

1997

The ability of Escherichia coli RNase H1 to hydrolyze structured substrates containing antisense oligonucleotides preannealed to a 47-mer RNA was compared with its ability to hydrolyze unstructured substrates containing antisense oligonucleotides duplexed with 13-mer RNA. These results demonstrate that when antisense oligonucleotides were bound to structured RNA, the resultant duplexes were cleaved at rates significantly slower than when the same oligonucleotides were bound to unstructured oligoribonucleotides. Structured substrates exhibited fewer cleavage sites, and each cleavage site was cleaved less rapidly than in unstructured substrates. Furthermore, the enzymatic activity of E. coli RNase H1 for the structured substrates was most affected when the cleavage sites corresponding to the enzymatically most active sites on the unstructured substrates were blocked in the structured substrates. Molecular modeling suggests that the observed ablation of RNase H activity was due to the steric hindrance of the enzyme by the structured RNA, i.e. steric interference of the phosphate groups on the substrate and/or the binding site of the enzyme. When chimeric oligonucleotides composed of a five-base deoxynucleotide sequence flanked by chemically modified nucleotides were bound to structured RNA, the resultant duplexes were even worse substrates for RNase H. These results offer further insights into the role of antisense-induced RNA structure on RNase H activity and may facilitate the design of effective antisense oligonucleotides.