A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29 -30 2-O-Me nucleotides, 1-2 other 2-modified nucleotides at positions U4 and U7, and a 3-3-linked nucleotide "cap." Eight 2-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2-NH 2 substitutions at U4 and U7, or 2-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only ϳ0.1 min. The addition of a 3-3-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000 -80,000-fold compared to the all-RNA parent ribozyme.Trans-acting ribozymes exert their activity in a highly specific manner and are therefore not expected to be detrimental to non-targeted cell functions. Because of this specificity, the concept of exploiting ribozymes for cleaving a specific target mRNA transcript is now emerging as a therapeutic strategy in human disease and agriculture (Cech, 1992;Bratty et al., 1993). For ribozymes to function as therapeutic agents, they may be introduced exogenously or produced endogenously in the target cells. In the former case, the chemically modified ribozyme must maintain its catalytic activity while also being stable to nucleases. A major advantage of chemically synthesized ribozymes is that site-specific modifications may be introduced at any position in the molecule. This approach provides flexibility in designing ribozymes that are catalytically active and stable to nucleases. In this manuscript we show that using this site-specific, chemical modification strategy, ribozymes can be designed that have wild-type catalytic activity and are not cleaved by nucleases.A variety of selective and uniform structural modifications have been applied to oligonucleotides to enhance nuclease resistance (Uhlmann and Peyman, 1990;Beaucage and Iyer, 1993;Milligan et al., 1993). Improvements in the chemical synthesis of RNA (Scaringe et al., 1990;Wincott et al., 1995) have led to the ability to similarly modify ribozymes containing the hammerhead ribozyme core motif Yang et al., 1992) (Fig. 1). Yang et al. (1992) demonstrated that 2Ј-O-Me modification of a ribozyme at all positions except G5, G8, A9, A15.1, and G15.2 (see numbering scheme in Fig. 1) led to a catalytically active molecule having a greatly decreased k cat value in vitro, but a 1000-fold increase in nuclease resistance over that of an all-RNA ribozyme when tested in a yeast extract. In another study (Paolella...

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A synthetic route to nucleoside 5'-deoxy-5'-difluoromethyl phosphonates from ribofuranosyl &deoxy-5-difluoromethyl phosphonate precursors is described. Methyl 5,6-dideoxy-6-(diethoxyphosphinyl)-6,6-difluoro-2,3-0-isopropylidene-fi-~-ribo-hexohranoside (7) was converted, under mild conditions, to the suitable glycosylating agent l-O-acetyl-2,3-di-O-benzoyl-5,6-dideoxyS-(diethoxyphosphinyl)-6,6-difluoro-fi-~-ribo-hexofuranoside (10). 1,2-Di-O-acetyl-3-O-benzyl-5,6-dideoxy-6-(dietho~hos-phinyl)-6,6-difluoro-fi-~-ribo-hexofuranoside (16) was also prepared as a versatile building block for nucleotide synthesis. Condensation of 10 with silylated nucleobases, followed by complete deprotection, afforded 5',6'-dideoxy-6'-(dihydroxyphosphinyl~-6',6'-difluoro nucleoside analogs 22a-c. In the case of the glycosylation of adenine, a considerable quantity of N-7 regioisomer 19 was formed. 5',6'-Dideoxy-6'-(dihydroxyphosphinyl)-6',6'-difluoro adenosine analog 22c was converted into the triphosphate analog 23 using 1,l'-carbonyldiimidazole activation followed by condensation with pyrophosphate. The adenosine 3',5'-cyclic monophosphate analog 24 was obtained through the DCC promoted intramolecular cyclization of 22c. Dinucleoside phosphate analog 27 was prepared by DCC-catalyzed coupling of l-[2,3-di-O-benzoyl-5,6-dideoxy-6-(dihydrox~hosphinyl)-6,6-difluoro-fi-~-ribo-hexofuranosylluraci1(21a) with 2',5'-bis(0-tert-butyldimethylsily1)-N4-acetylcytidine (263, followed by deprotection.

A facile synthetic route for the 4′-thioribonucleoside building block 4′SN (N = U, C, A and G) with the ribose O4′ replaced by sulfur is presented. Conversion of l-lyxose to 1,5-di-O-acetyl-2,3-di-O-benzoyl-4-thio-d-ribofuranose was achieved via an efficient four-step synthesis with high yield. Conversion of the thiosugar into the four ribonucleoside phosphoramidite building blocks was accomplished with additional four steps in each case. Incorporation of 4′-thiocytidines into oligoribonucleotides improved the thermal stability of the corresponding duplexes by ∼1°C per modification, irrespective of whether the strand contained a single modification or a consecutive stretch of 4′SC residues. The gain in thermodynamic stability is comparable to that observed with oligoribonucleotides containing 2′-O-methylated residues. To establish potential conformational changes in RNA as a result of the 4′-thio modification and to better understand the origins of the observed stability changes, the crystal structure of the oligonucleotide 5′-r(CC4′SCCGGGG) was determined and analyzed using the previously solved structure of the native RNA octamer as a reference. The two 4′-thioriboses adopt conformations that are very similar to the C3′-endo pucker observed for the corresponding sugars in the native duplex. Subtle changes in the local geometry of the modified duplex are mostly due to the larger radius of sulfur compared to oxygen or appear to be lattice-induced. The significantly increased RNA affinity of 4′-thio-modified RNA relative to RNA, and the relatively minor conformational changes caused by the modification render this nucleic acid analog an interesting candidate for in vitro and in vivo applications, including use in RNA interference (RNAi), antisense, ribozyme, decoy and aptamer technologies.

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