Novel RNA Synthesis Method Using 5‘-O-Silyl-2‘-O-orthoester Protecting Groups

Abstract

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 conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

“…RNA strands representing the regions of the U2 snRNA and intron involved in the complementary branch-site interaction were chemically synthesized as short duplexes (sequences shown in Fig+ 1C)+ Oligomers representing the U2 snRNA fragment (GGU GUA GUA and GGU GdcA GUA), where dc is deoxypseudouridine, were paired with a complementary region of the intron strand (UAC UAA CAC C, where the underlined A corresponds to the branch site)+ An oligomer without the branch-site A (UAC UAC ACC, which formed an unbulged duplex when paired with the U2 strand) was also synthesized+ Oligomers were synthesized trityl-off on a converted ABI model 430A peptide synthesizer using standard phosphoramidite chemistry+ RNA oligomers were synthesized on a 5 mmol scale with commercial phosphoramidites on 500 Å CPG solid support resin (GLEN Research, Sterling, Virginia), using adapted DNA synthesis cycles and 720-s coupling times with 0+05-M RNA phosphoramidite solutions+ Oligomers were cleaved from solid support and deprotected according to the manufacturer's protocol and lyophilized to dryness+ Triethylamine trihydrofluoride (Sigma) was added to the dried RNA at 1 mL per micromole to desilylate the 29OH groups+ Reactions were quenched after 24 h with 300 mL of water per micromole and the RNA was precipitated with n-butanol (Westman & Stromberg, 1994)+ The top strand sequence of cBP (GGU GcA GUA), purchased from Dharmacon Research, Inc+ (Boulder, Colorado), was synthesized using 59-silyl-29-orthoester oligonucleotide synthesis chemistry (Meroueh et al+, 2000;Scaringe, 2000) and deprotected according to company protocols for ACE chemistry (Scaringe et al+, 1998)+ RNA oligomers were purified by HPLC using a DNAPac PA-100 semi-prep anion exchange column (Dionex, Inc+), with a 20 mM Tris, pH 8 mobile phase+ RNA oligomers were eluted with a 500-mM NaClO 4 gradient of 1% per minute+ Recovered fractions were concentrated using a stirred-cell apparatus (Amicon, Inc+) with a 1000 MWCO membrane+ Concentrated fractions were washed with a high salt buffer (1 M NaCl, 10 mM Na phosphate, 0+1 mM EDTA, pH 6+4) to exchange out residual Tris ions+ The samples were then exchanged into a low salt buffer (50 mM NaCl, 10 mM Na phosphate, 0+1 mM EDTA, pH 6+4)+ Equimolar amounts of top and bottom strands were combined to form duplexes+ The samples were dried and resuspended in 90% H 2 O/10% 2 H 2 O (Cambridge Isotope Laboratories) for NMR observation of exchangeable protons, or were dried twice from 99+9% 2 H 2 O, once from 99+96% 2 H 2 O, and finally suspended in 99+96% 2 H 2 O for NMR studies of nonexchangeable protons+ Sample concentrations were in the range of 0+8 mM to 1+2 mM at a volume of approximately 250 mL+ Microvolume NMR tubes (Shigemi, Inc+) were used for NMR data collection+ MgCl 2 (hexahydrate, SigmaUltra) was added to a final concentration of 5 mM in NMR samples of uBP and cBP+ Magnesium was removed by adding 10 mM EDTA to the NMR samples and exchanging into low salt buffer again+ Cobaltic hexamine chloride (Acros, Inc+) was then added to a concentration of 5 mM for subsequent studies+ The pH of uBP and cBP was adjusted using 2-mL aliquots of 0+5 M HCl or 0+5 M NaOH and measuring pH changes with a microelectrode (Ingold, Inc+)+ Thermal denaturation studies UV absorbance measurements were performed on a Cary 3E spectrophotometer with temperature control using Varian Cary WinUV software+ Dry nitrogen gas was flowed through the cell block to prevent condensation below the atmospheric dew point+ Temperature was varied from 10 8C to 80 8C at a rate of 1 8C per min, and absorbance measurements at 260 nm were recorded at each integer temperature inter...…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…Chemical synthesis is the method of choice for preparing small RNAs, as in vitro enzymatic synthesis of RNAs smaller than 10 nucleotides has been reported not to be successful [18], except in one case [30]. Chemical synthesis of RNA is reported for RNAs up to 80 nucleotides [92][93][94]. However, low yields and high costs for larger RNAs make the chemical synthesis suitable only for short RNAs (<20nt).…”

Combined Approaches to Site-Specific Modification of RNA

“…Some examples include protecting groups that are fluoride-labile (e.g., TBDMS) (19), acid labile (e.g., Fpmp) (20), photolabile (e.g., Nbn) (8) or that are removed under reducing conditions (e.g., DTM) (21) or by metal catalysis (e.g., allyl) (22,23) ( Table 1). (27). Because of the presence of the silyl 5′-protecting group, which requires fluoride deprotection in each cycle, the synthesis is carried out on polystyrene (PS) supports rather than silica-based supports (e.g., CPG).…”

“…In 1998, Scaringe and coworkers developed a different strategy for RNA chemical synthesis, which employs novel 5′-and 2′-O-protecting groups. Their approach utilizes 5′-O-DOD ether and 2′-O-ACE orthoester protecting groups along with the 3′-O-(methyl-N, N-diisopropyl) phosphoramidite (Figure 3, panel c) that gives coupling yields >99% (27). Because of the presence of the silyl 5′-protecting group, which requires fluoride deprotection in each cycle, the synthesis is carried out on polystyrene (PS) supports rather than silica-based supports (e.g., CPG).…”