Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.
The ability to routinely synthesize RNA has become increasingly important as research reveals the multitude of RNA's biological functions. 1 Over the past 25 years, many chemical strategies have been explored for synthesizing RNA. Most approaches have focused on retaining the 5′-O-dimethoxytrityl (DMT) ether and adding a compatible 2′-hydroxyl protecting group such as fluoride-labile silyl ethers, 2 photolabile moieties, 3 or acid-labile acetals. 4 The acetals have exhibited many attractive features, but a delicate balance has been required to successfully utilize the 2′-O-acetals and the 5′-O-DMT ether in the same synthesis strategy. 5 Hence, other approaches have involved retaining the 2′-O-acetal while replacing the 5′-O-DMT. 6 Several reviews further document these strategies. 7 Of all of the RNA synthesis methods reported to date, the 5′-O-DMT-2′-O-tertbutyldimethylsilyl (TBDMS) and the 5′-O-DMT-2′-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP) chemistries are offered commercially. Unfortunately, neither allows RNA synthesis to be as routine and dependable as DNA. The current methods enable the synthesis of RNA in acceptable yields and quality, but a high level of skill appears to be required to deliver adequate results. The need and desire exists for more robust RNA synthesis methods which consistently produce higher quality RNA.Whereas most previous approaches were adaptations of DNA methodologies, we focused on a de noVo strategy and asked what would be optimal for RNA. According to the literature, the most desirable conditions for the final 2′-O-deprotection would be mildly acidic aqueous conditions. The obstacle to using mildly acid-labile 2′-O-groups has been the 5′-O-DMT group, which is removed under similar conditions. Our investigations led to the successful development of silyl ethers for protection of the 5′-hydroxyl. 8 These protecting groups can be removed with fluoride ions under neutral conditions which are compatible with an acidlabile 2′-hydroxyl moiety. However, it was subsequently discovered that 5′-O-silyl ether oligonucleotide synthesis chemistry in conjunction with 2′-O-acetals produced side products. 8 Acidlabile orthoester protecting groups 9 were investigated as alternatives and discovered to be suitable for the 2′-hydroxyl.We recently developed the 2′-O-bis(2-acetoxyethoxy)methyl (ACE) orthoester that is stable to nucleoside and oligonucleotide synthesis conditions but is modified via ester hydrolysis during base deprotection of the oligonucleotide. 10 The resulting 2′-Obis(2-hydroxyethoxy)methyl orthoester is 10 times more acidlabile than the ACE orthoester. Complete cleavage of the 2′-Oprotecting groups is effected using extremely mild conditions (pH 3, 10 min., 55°C). The innovative features of this chemistry have enabled the synthesis of RNA oligonucleotides of unprecedented quality.The structures of the four RNA nucleoside phosphoramidites are illustrated in Figure 1. The 3′-hydroxyl was functionalized as the methoxy N,N-diisopropylphosphoramidite. (We observed that th...
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...
Kinetic analysis, using substrates that consisted entirely of deoxynucleotides with the exception of the single mandatory ribonucleotide at the cleavage site which contained either a 5‘-oxy- or 5‘-thio-leaving group, demonstrated that the departure of the 5‘-leaving group was not the rate-limiting step of a hammerhead ribozyme-catalyzed reaction [Kuimelis, R. G.; McLaughlin, L. W. J. Am. Chem. Soc. 1995, 117, 11019−11020]. We recently synthesized a natural all-RNA substrate that contains a 5‘-thio-leaving group at the cleavage site and performed detailed kinetic analysis. In contrast to the conclusion of Kuimelis and McLaughlin, we found that (i) the attack by the 2‘-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5‘-thio group (R11S) and (ii) the departure of the 5‘-leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both enzymatic and nonenzymatic reactions.
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