2′-O-MODIFIED OLIGORIBONUCLEOTIDES WITH TERMINAL 3′-3′-INTERNUCLEOTIDE LINKAGE AND THEIR DERIVATIVES

A high RNA binding affinity and nuclease resistance of 2′-O-modified (2′-O-methyl, 2′-O-tetrahydropyranyl) oligoribonucleotides containing the “inverted” T at the 3′-end have been shown. The synthesis and properties of new photoactivatable perfluoroarylazide derivatives of these oligoribonucleotides are discussed.     

A new method for 3''-labelling of polyribonucleotides by phosphorylation with RNA ligase and its application to the 3''-modification for joining reactions.

P1-Adenosine 5'-P2-o-nitrobenzyl pyrophosphate (nbzlppA) has been synthesized as a substrate for T4 RNA ligase catalyzed 3'-phosphorylation. Incubation of oligoribonucleotides and nbzlppA with RNA ligase yielded oligoribonucleotides having a 3'-o-(o-nitrobenzyl) phosphate. Photochemical removal of the o-nitrobenzyl group provided the free 3'-phosphate. Using [P2-32P] nbzlppA, 3'-termini of oligoribonucleotides could be labelled with 32P. This reaction was applied to modify the 3'-end of donor molecules in joining reaction with RNA ligase. A trinucleotide U-A-G was converted to U-A-Gpnbzl and phosphorylated with polynucleotide kinase. pU-A-Gpnbzl was then joined to an acceptor trinucleotide A-U-G to yield A-U-G-U-A-Gp.     

Conformational and stacking properties of 3′–5′ and 2′–5′ linked oligoribonucleotides studied by CD

Comparative CD studies have been carried out to characterize the properties of 2′–5′ and 3′–5′ oligoriboadenylates and oligoribouridylates from dimer to decamer. The CD band of the 3′–5′ oligoribonucleotides was larger than that of the 2′–5′ oligoribonucleotides and increased with the increase in chain length, while the CD band of the 2′–5′ oligoribonucleotides increased little beyond the dimer level. The CD analysis of the chain length dependency revealed that the 3′–5′ oligoribonucleotides adopt mainly the base-base stacking interaction, while the base-sugar interaction is predominant in the 2′–5′ oligoribonucleotides. The CD intensity of 3′–5′ oligoribonucleotides decreased to a larger extent at elevated temperatures or in the presence of ethanol compared to that of the 2′–5′ counterparts. Mg²⁺ or Mn²⁺ ion enhanced the magnitude of the CD of 3′–5′ octariboadenylate, while a small decrease in the CD was observed by the presence of Mg²⁺ or Mn²⁺ ion to the 2′–5′ octariboadenylate. The 3′–5′ oligoribonucleotide is likely conformationally flexible and can form helical ordered structure with strong base-base stacking depending on changes in the environment such as temperature, the presence of Mg²⁺ ion, or hydrophobicity of the solution. © 1996 John Wiley & Sons, Inc.     

Sequence-selective cleavage of oligoribonucleotides by 3d transition metal complexes of 1,5,9-triazacyclododecane-functionalized 2'-O-methyl oligoribonucleotides

2'-O-Methyl oligoribonucleotides bearing a 3'-[2,6-dioxo-3,7-diaza-10-(1,5,9-triazacyclododec-3-yl)decyl phospate conjugate group have been shown to cleave in slight excess of Zn(2+) ions complementary oligoribonucleotides at the 5'-side of the last base-paired nucleotide. The cleavage obeys first-order kinetics and exhibits turnover. The acceleration compared to the monomeric Zn(2+) 1,5,9-triazacyclododecane chelate is more than 100-fold. In addition, 2'-O-methyl oligoribonucleotides having the 1,5,9-triazacyclododec-3-yl group tethered to the anomeric carbon of an intrachain 2-deoxy-beta-d-erythro-pentofuranosyl group via a 2-oxo-3-azahexyl, 2,6-dioxo-3,7-diazadecyl, or 2,9-dioxo-3,10-diazatridecyl linker have been studied as cleaving agents. These cleave as zinc chelates a tri- and pentaadenyl bulge opposite to the conjugate group approximately 50 times as fast as the monomeric chelate and show turnover. The cleavage rate is rather insensitive to the length of linker. Interestingly, a triuridyl bulge remains virtually intact in striking contrast to a triadenyl bulge. Evidently binding of the zinc chelate to a uracil base prevents its catalytic action. Replacement of Zn(2+) with Cu(2+) or Ni(2+) retards the cleaving activity of all the cleaving agents tested.     

The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2'-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2'-O-phosphate)-3'-phosphoramidites, and a new condition for removal of 2'-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2'-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2'-phosphotransferase.     

The Chemical Synthesis of Oligoribonucleotides with Selectively Placed 2′-O-Phosphates

Abstract

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2′-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2′-O-phosphate)-3′-phosphoramidites, and a new condition for removal of 2′-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2′-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2′-phosphotransferase.     

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.     

Perfluoroarylazide derivatives of 2'-O-modified oligoribonucleotides: efficient reagents for RNA photomodification

Site-specific photomodification of the 5'-terminal fragment of MDR1 mRNA by perfluoroarylazide derivatives of 2'-O-modified (2'-O-methyl or 2'-O-tetrahydropyranyl) oligoribo- and oligodeoxyribonucleotides was investigated. The conjugates built of 2'-O-modified oligoribonucleotides demonstrate beneficial features compared with their deoxyribo analogs: the extent of RNA modification by 2'-O-modified oligoribonucleotides and oligodeoxyribonucleotide conjugates was 40- 50% and 20%, respectively.     

Synthesis of oligoribonucleotides via ribonucleoside-H-phosphonates

The synthesis of oligoribonucleotides via 5'-O-dimethoxytrityl-2'-E-tetrahydropyranyl-N-acylribonucleoside 3'-H-phosphonates is described. With these synthones and pivaloyl chloride as condensing agent, the synthesis of CpC and GpA in solution and the polymer support synthesis of (Up)4U and (Up)5C in manual variant and at the automatic synthesizer "Victoria-4M" was performed. The obtained oligonucleotide-H-phosphonates were oxidized to phosphates both with usually employed aqueous iodine solution and with of CCl4-Et3N-H2O (the Todd-Atherton reaction).     

New methodology for the synthesis of 2'(3')-O-aminoacyl oligoribonucleotides related to the 3'-terminus of aa-tRNA

A new synthetic approach to the title compounds is reported. It is based on the phosphotriester methodology and uses a unique combination of protecting groups (Fmoc and Ph for the aglycons; DMT, CPTr and Mthp for the carbohydrate hydroxy functions; 2-CIPh for the phosphodiester bonds; BPOC for amino acid). This enables selective deprotection of the blocked oligonucleotide intermediates in two steps (oximate and acid treatments) to yield the 2'(3')-O-aminoacyl oligoribonucleotides suitable for biochemical investigations.     

Synthesis of 2′-O-Photocaged Ribonucleoside Phosphoramidites

The chemical synthesis and incorporation of the phosphoramidite derivatives of 2?′-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging of these 2′-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl < 4,5-dimethoxy-2-nitrobenzyl < 2′-O-o-nitrobenzyl.     

Helical Structure Formation Between Complementary Oligonucleotides. Minimum Chain Length Required for the Template-Directed Synthesis of Oligonucleotides

Helix formation between various combinations of 3–5 linked oligoribouridylates and oligoriboadenylates from dimer to dodecamer has been studied to gain information on the chain-length requirement for the template-directed condensation of oligoribonucleotides. We have measured the helix formation under high oligoribonucleotide concentration in the presence of magnesium ion at 0–50°C by UV or CD, as many model processes of oligoribonucleotides replication have been carried out under such conditions. Adenylic acid, (pA), diadenylic acid, (pA)₂, or triadenylic acid, (pA)₃, forms a helix with poly(U) or oligo(U) with a chain length of more than eight. On the other hand, neither uridylic acid, (pU), nor diuridylic acid, (pU)₂, can form a helix with oligo(A) or poly(A). Triuridylic acid, (pU)₃, or the longer oligo(U) forms a helix with oligo(A) with a chain length of over six. The results suggest that a trimer is the minimum unit as an incorporating nucleotide for conducting any set of nonenzymatic template-directed synthesis, AU and UA, as the nonenzymatic template-directed condensation of oligoribonucleotides correlates well with the results of helix formation of complementary oligoribonucleotides. We have further found the partial helix formation between 2–5 linked decauridylate, (pU)₁₀, and pA or 2–5 linked (pA)₂ at 0 °C, which indicates the possibility of the template activity of long 2–5 linked oligonucleotides for the nonenzymatic oligonucleotide synthesis.     

Cleavage of synthetic RNA-DNA hybrids with restriction endonucleases BamH1 and Sau3A

Synthetic oligodeoxyribonucleotides, containing one or two ribonucleotides in the recognition sequence, and RNA--DNA hybrids were tested for their activity in cleavage with BamH1 and Sau3A endonucleases. The replacement of dG with G in the first position of BamH1-site (GGATCC) of one of the chains does not affect the rate of the BamH1 hydrolysis. The similar heteroduplex, containing G residue in the second position, displays a decreased rate of the BamH1 hydrolysis of the modified strand and, to a lesser extent, of the unmodified complementary strand. Oligodeoxyribonucleotides in complex with oligoribonucleotides can be cleaved with the excess of BamH1 and Sau3A, oligoribonucleotides remaining intact.     

Synthesis and characterization of a ribavirin-3'',5''-phosphate pentadecamer homoribopolymer bearing a 5''-amino tether group and a 3''-thymidine.

The synthesis of a pentadecamer of the 5'-phosphate of the antiviral nucleoside ribavirin (1'-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) has been achieved. This homoribopolymer is terminated at the 5'-position with an (6-aminohexyl)phosphate group to permit conjugation to a carrier and at the 3'-position by a thymidine-5'-phosphate. The synthesis was accomplished using the methyl phosphoramidite approach to oligoribonucleotides. The homoribopolymer was insensitive to ribonuclease A but was sensitive to ribonuclease T2 digestion.     

An RNA microchip containing immobilized oligoribonucleotides with protective groups at 2'-O-positions

To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions.     

Solid phase synthesis of 5'-diphosphorylated oligoribonucleotides and their conversion to capped m7Gppp-oligoribonucleotides for use as primers for influenza A virus RNA polymerase in vitro.

We have synthesized four different 5'-diphosphorylated oligoribonucleotides, varying in length from 11 to 13 nucleotides by a new solid phase method. After deprotection and partial purification the 5'-diphosphorylated oligoribonucleotides could be converted to capped (m7Gppp) oligoribonucleotides using guanylyl transferase. Radiolabelled capped oligoribonucleotides acted as primers for the influenza A virus RNA polymerase in vitro. The solid phase method described here should also allow the addition of 5'-diphosphates to synthetic oligodeoxyribonucleotides and be capable automation.     

A rapid method for purification of synthetic oligoribonucleotides.

A procedure for large-scale purification of synthetic oligoribonucleotides has been developed that has significant advantages over gel purification techniques currently in use. Synthesis was performed using commercially available 2'-O-silylated ribonucleoside 3'-O-phosphoramidites, and coupling efficiencies were consistently greater than 97% for oligoribonucleotides up to 31 residues in length. Using C4 reverse-phase chromatography to remove material not deprotected by treatment with tetrabutylammonium fluoride, we have eliminated reactants in which the 2'-O-silyl group is only partly removed, thus ensuring a homogeneous population of oligoribonucleotide.     

The Protection of The 2′-Hydroxyl Function in Oligoribonucleotide Synthesis

Abstract

A new, easily accessible and achiral 2′-ketal protective group has been designed for the use in the chemical synthesis of oligoribonucleotides; the proposed 2′-ketal group⁽¹⁾ has the additional advantage that it could be easily functionalized to the diamide (6) with aq. ammonia at the penultimate step of deblocking of oligoribonucleotides which makes it more acid-labile than the parent 2′-ketal group during the final acid-promoted deprotection step.     

Phosphoryl migration during the chemical synthesis of RNA.

By the use of high sensitivity assay systems, we have measured the occurrence of strand scission and phosphoryl migration that accompany the deblocking of chemically synthesized oligoribonucleotides. Substantial phosphoryl migration was observed for both enzymatically derived poly(uridylic acid) and synthetic uridine oligoribonucleotides 2'-O-protected with the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) group, when these species were subjected to the acidic conditions suggested for Fpmp deprotection. Strand scission occurred in parallel and could be demonstrated readily by 5'-32P end labeling, but not by 3'-32P end labeling, of the acid-treated oligoribonucleotides. Increasing the pH of the deprotection solution and decreasing the temperature at which the deprotection was accomplished diminished both phosphoryl migration and strand scission. A mechanism that can rationalize these results is discussed.     

Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), beta-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic 'Hammerhead Ribozyme'. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.