RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds

Abstract

Three types of 14-mer oligonucleotides were hybridized to human beta-globin pre-mRNA and the resultant duplexes were tested for susceptibility to cleavage by RNase H from E. coli or from HeLa cell nuclear extract. The oligonucleotides contained normal deoxynucleotides, phosphorothioate analogs alternating with normal deoxynucleotides, or one to six methylphosphonate deoxynucleosides. Duplexes formed with deoxyoligonucleotides or phosphorothioate analogs were susceptible to cleavage by RNase H from both sources, whereas a duplex formed with an oligonucleotide containing six methylphosphonate deoxynucleosides alternating with normal deoxynucleotides was resistant. Susceptibility to cleavage by RNase H increased parallel to a reduction in the number of methylphosphonate residues in the oligonucleotide. Stability of the oligonucleotides in the nuclear extract from HeLa cells was also tested. Whereas deoxyoligonucleotides were rapidly degraded, oligonucleotides containing alternating methylphosphonate residues remained unchanged after 70 minutes of incubation. Other oligonucleotides exhibited intermediate stability.

Stereodifferentiation—the effect of P chirality of oligo (nucleoside phosphorothioates) on the activity of bacterial RNase H

“…PS-oligos which form duplexes with RNA that are recognized by RNase H (9) are considered the most promising potential therapeutics to date. The selection is based on the close resemblance of PS-oligos to the natural oligonucleotides (isosteric and isoelectronic with natural oligonucleotides), their known resistance to nucleolytic enzymes (10) and their ability to activate RNase H. Our research is focused on the effective chemical synthesis of PS-oligos (11)(12)(13)) and the precise description oftheir structure and diastereomeric composition (14).…”

Potential roles of antisense technology in cancer chemotherapy

“…In contrast, phosphorothioates are excellent substrates (Cazenave et al, 1989;Mirabelli et al, 1991;Stein and Cheng, 1993). In addition, chimeric molecules have been studied as oligonucleotides that bind to RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989). For example, oligonucleotides comprised of wings of 2'-methoxy phosphonates and a ®ve-base gap of oligodeoxynucleotides bind to their target RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989).…”

“…In addition, chimeric molecules have been studied as oligonucleotides that bind to RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989). For example, oligonucleotides comprised of wings of 2'-methoxy phosphonates and a ®ve-base gap of oligodeoxynucleotides bind to their target RNA and activate RNaseH (Furdon et al, 1989;Quartin et al, 1989). Furthermore, a single ribonucleotide in a sequence of deoxyribonucleotides was shown to be su cient to serve as a substrate for RNaseH when bound to its complementary deoxyoligonucleotide (Eder and Walder, 1991).…”

RNase H is responsible for the non-specific inhibition ofin vitrotranslation by 2′-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers

“…This is the case with methylphosphonates (3,4), phosphoramidates (5), 2'-O-alkyl RNA (6) and alpha analogues (4,7,8). Composite oligonucleotides made of differently modified (or unmodified) parts were prepared that aimed at combining different properties within a single molecule (9)(10)(11). Mixed methylphosphonate-phosphodiester oligonucleotides were synthesized in order to yield antisense sequences resistant to exonucleases and nevertheless able to elicit RNase H activity once bound to their RNA target (10,12).…”

“…Composite oligonucleotides made of differently modified (or unmodified) parts were prepared that aimed at combining different properties within a single molecule (9)(10)(11). Mixed methylphosphonate-phosphodiester oligonucleotides were synthesized in order to yield antisense sequences resistant to exonucleases and nevertheless able to elicit RNase H activity once bound to their RNA target (10,12). Phosphoramidate-phosphorothioate sandwich oligomers were also used against An2 cyclin mRNA in injectedXenopus embryos (13).…”