Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA

The synthesis of a 5′-O-BzH–2′-O-ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G₁₉₀₆–C₁₉₂₄) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudouridine at the stem–loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.     

An automated procedure for the analysis of methylated nucleosides in nuclear RNA and mRNA.

A commercial high pressure liquid chromatographic system is used for the rapid analysis of 2′-O-methylnucleosides and pyrophosphate-linked methylated termini of nuclear RNAs and mRNAs. Oligonucleotides or RNA samples were enzymatically hydrolyzed to nucleosides and analyzed automatically by cationexchange chromatography in less than 1 hr using a 0.4 m ammonium formate buffer system at 55° c. This method is sufficiently sensitive to detect the N⁷-methylguanosine residue in 0.1 mg of Novikoff ascites hepatoma mRNA and may be used for qualitative or quantitative studies on RNA methylation in vivo or in vitro.     

Synthesis and evaluation against hepatitis C virus of 7-deaza analogues of 2′-C-methyl-6-O-methyl guanosine nucleoside and l-Alanine ester phosphoramidates

7-Deazapurines are known to possess broad antiviral activity, however the 2′-C-methylguanosine analogue displays poor cell permeation and limited phosphorylation, thus is not an efficient inhibitor of hepatitis C virus (HCV) replication. We previously reported the 6-O-methyl entity as a prodrug moiety to increase liphophilicity of guanine nucleosides and the ProTide approach applied to 2′-C-methyl-6-O-methylguanosine has lead to potent HCV inhibitors now in clinical trials. In this Letter, we report the synthesis and biological evaluation of 2′-C-methyl-6-O-methyl-7-deaza guanosine and ProTide derivatives. In contrast to prior studies, removal of the N-7 of the nucleobase entirely negates anti-HCV activity compared to the 2′-C-methyl-6-O-methylguanosine analogues. To understand better this significant loss of activity, enzymatic assays and molecular modeling were carried out and suggested 2′-C-methyl-6-O-methyl-7-deaza guanosine and related ProTides do not act as efficient prodrugs of the free nucleotide, in marked contrast to the case of the parent guanine analogue.     

Synthesis of the Oligoribonucleotides Incorporating 8-Oxo-Guanosine and Evaluation of their Base Pairing Properties

6-O-7-N-Bis(diphenylcarbamoyl)-2-N-phenoxyacetyl-5′-O-dimethoxytrityl-2′-O-{[(triisopropyl- silyl)oxy]methyl}-8-oxoguanosine-3′-yl-β-cyanoethyl-N,N-diisopropylphosphoramidite (5) was synt- hesized as a new phosphoramidite precursor unit for the synthesis of RNA. Compound 5 was successfully incorporated into the middle of the RNA sequences, and the synthesized RNAs were identified by MALDI-TOF mass measurements. Their properties were evaluated for formation of the RNA duplex and RNA/DNA heteroduplex. ORNs 1 and 4 containing 8-oxo-G can form base pairs with rC or dC in an anti conformation, while it can also interact with rA or dA in a syn conformation in the RNA duplex or RNA/DNA heteroduplex. The described synthetic method is therefore a useful procedure for the synthesis of ORN containing 8-oxo-G and contributes to the study of 8-oxo-G in RNA.     

Synthesis of guanosine and its derivatives from AICA-riboside

A novel and convenient method for the synthesis of guanosine is described. The reaction of AICA-riboside with sodium methylxanthate gave 2-mercaptoinosine in almost quantitative yield. The latter was oxidized with hydrogen peroxide to afford inosine-2-sulfonic acids, which was readily animated to give guanosine in excellent yield. Similarly, the preparation of N²-methylguanosine and N²,N²-dimethylguanosine, minor constituents of transfer RNA, was also accomplished. Furthermore, this procedure was extended to the synthesis of 2′,3′-O-isopropylideneguanosine and the isopropylidene derivatives of various N²-substituted guanosines from 2′,3′-O-isopropylidene-AICA-riboside. Guanosine via 2′,3′-O-isopropylideneguanosine was successfully phosphorylated to give 5′-guanylic acid.     

Mapping messenger RNA methylations at single base resolution

The messenger RNA (mRNA) methylations in mammalian cells have been found to contain N⁶-methyladenosine (m⁶A), N⁶-2′-O-dimethyladenosine (m⁶Am), 7-methylguanosine (m⁷G), 1-methyladenosine (m¹A), 5-methylcytosine (m⁵C), and 2′-O-methylation (2′-OMe). Their regulatory functions in control of mRNA fate and gene expression are being increasingly uncovered. To unambiguously understand the critical roles of mRNA methylations in physiological and pathological processes, mapping these methylations at single base resolution is highly required. Here, we will review the progresses made in methylation sequencing methodologies developed mainly in recent two years, with an emphasis on chemical labeling-assisted single base resolution methods, and discuss the problems and prospects as well.     

Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion

We have systematically investigated the duplex to hairpin conversion of oligoribonucleotides under the aspect of nucleobase methylation. The first part of our study refers to the self-complementary sequence rCGCGAAUUCGCGA, which forms a stable Watson–Crick base paired duplex under various buffer conditions. It is shown that this sequence is forced to adopt a hairpin conformation if one of the central 6 nt is replaced by the corresponding methylated nucleotide, such as 1-methylguanosine N²,N²-dimethylguanosine, N⁶,N⁶-dimethyladenosine (m⁶₂A) or 3-methyluridine. On the other hand, the duplex structure is retained and even stabilized by replacement of a central nucleotide with N²-methylguanosine (m²G) or N⁴-methylcytidine. A borderline case is represented by N⁶-methyladenosine (m⁶A). Although generally a duplex-preserving modification, our data indicate that m⁶A in specific strand positions and at low strand concentrations is able to effectuate duplex–hairpin conversion. Our studies also include the ssu ribosomal helix 45 sequence motif, rGACCm²GGm⁶₂Am⁶₂AGGUC. In analogy, it is demonstrated that the tandem m⁶₂A nucleobases of this oligoribonucleotide prevent duplex formation with complementary strands. Therefore, it can be concluded that nucleobase methylations at the Watson–Crick base pairing site provide the potential not only to modulate but to substantially affect RNA structure by formation of different secondary structure motifs.     

2��-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m⁷GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N⁶-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.     

Facile and efficient approach for the synthesis of N2-dimethylaminomethylene-2′-O-methylguanosine

A convenient method for the synthesis of N²-dimethylaminomethylene-2′-O-methylguanosine (1), which is a useful intermediate for oligonucleotide construction, was developed. We chose the di-tert-butylsilyl group and the triisopropylbenzenesulfonyl group as sugar and base protecting groups, respectively. These protecting groups were stable during the 2′-O-methylation step with MeI and NaH. Our six-step synthesis of 1 is easy to perform using commercially available reagents, and requires only three chromatographic purifications. Compound 1 was obtained in 56% yield from guanosine.     

Effective gene silencing activity of prodrug-type 2′-O-methyldithiomethyl siRNA compared with non-prodrug-type 2′-O-methyl siRNA

Small interfering RNAs (siRNAs) are an active agent to induce gene silencing and they have been studied for becoming a biological and therapeutic tool. Various 2′-O-modified RNAs have been extensively studied to improve the nuclease resistance. However, the 2′-O-modified siRNA activities were often decreased by modification, since the bulky 2′-O-modifications inhibit to form a RNA-induced silencing complex (RISC). We developed novel prodrug-type 2′-O-methyldithiomethyl (MDTM) siRNA, which is converted into natural siRNA in an intracellular reducing environment. Prodrug-type 2′-O-MDTM siRNAs modified at the 5′-end side including 5′-end nucleotide and the seed region of the antisense strand exhibited much stronger gene silencing effect than non-prodrug-type 2′-O-methyl (2′-O-Me) siRNAs. Furthermore, the resistances for nuclease digestion of siRNAs were actually enhanced by 2′-O-MDTM modifications. Our results indicate that 2′-O-MDTM modifications improve the stability of siRNA in serum and they are able to be introduced at any positions of siRNA.     

Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological properties

In this paper, we describe the synthesis of 4′-C-aminoalkyl-2′-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4′-C-aminoethyl and 4′-C-aminopropyl-2′-O-methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4′-C-aminoalkyl-2′-O-methyluridine adopts an C2′-endo sugar puckering in dsRNA. The 4′-C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules.     

Synthesis and Properties of Phosphorylated 3′-O-β-D-Ribofuranosyl-2′-deoxythymidine

Abstract

A strategy was developed for the synthesis of 3′-O-β-D-ribofuranosyl 2′-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5′-O- and 5″-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy.     

Synthesis of Modified Nucleotide Building Blocks Containing Electrophilic Groups in the 2′-Position

Abstract

Chemical syntheses of 2′-O-(allyloxycarbonyl)methyladenosine, 2′-O-(methoxycarbonyl)methyladenosine and 2′-O-(2,3-dibenzoyloxy)propyluridine 3′-2-cyanoethyl-N,N-diisopropyl phosphoramidite building blocks are described. These monomers were used successfully to incorporate carboxylic acid, 1,2-diol and aldehyde functionalities into synthetic oligonucleotides.     

New syntheses of 2′-C-methylnucleosides starting from d-glucose and d-ribose

Effective general methods have been developed for the synthesis of 2′-C-methylnucleosides starting from d-glucose and d-ribose. 3-O-benzyl-1,2-O-isopropylidene-3-C-methyl-α-d-allofuranose was prepared in 5 steps from d-glucose and converted into 1,2,3-tri-O-acetyl-2-C-methyl-5-O-p-methylbenzoyl-d-ribofuranose (5), the starting compound for nucleoside synthesis. Compound 5 was also synthesised from 2-C-hydroxymethyl-2,3-O-isopropylidene-5-O-trityl-d-ribofuranose, prepared in 3 steps from d-ribose. Condensation of 5 with the bis-trimethylsilyl derivatives of uracil, N⁴-benzoylcytosine, and N⁶-benzoyladenine in the presence of F₃CSO₃OSiMe₃ followed by removal of the protecting acyl groups yielded the corresponding 2′-C-methylnucleosides.     

Synthesis of the allo-analogue of trehalose

Selective benzoylation of HO-2 and HO-2′ of 4,6-O-benzylidene-α-D-glucopyranosyl 4,6-O-benzylidene-α-D-glucopyranoside with N-benzoylimidazole led to the exclusive formation of 2-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranosyl 2-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside. Oxidation of either the dibenzoate or the corresponding ditosylate with methyl sulphoxide-phosphorus pentaoxide gave the 3,3′-diulose, and subsequent reduction with borohydride gave the 3,3′diepimers having the allo-allo configuration. De-esterification and hydrolysis of the benzylidene substituents gave α-D-allopyranosyl α-D-allopyranoside.     

Preliminary SAR analysis of novel antiproliferative N6,5′-bis-ureidoadenosine derivatives

A preliminary library of novel N⁶,5′-bis-ureidoadenosine analogs and related derivatives was prepared and tested for activity against the NCI 60 panel of human cancers. A 2′-O-TBS group was found to be necessary, but not sufficient, for optimal antiproliferative activity. Neither the N⁶- nor 5′-ureido substituents were sufficient to achieve significant antiproliferative effects when present in the absence of the other. The 2′-O-TBS, and N⁶,5′-bis-ureido substitution patterns were found to be necessary for optimal antiproliferative activity.     

Improved Targeting of the Flanks of a DNA Stem Using α-Oligodeoxynucleotides.-The Enhanced Effect of an Intercalator

Abstract

2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N ⁴-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N ⁴-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region.

     

Syntheses of prodrug-type 2′-O-methyldithiomethyl oligonucleotides modified at natural four nucleoside residues and their conversions into natural 2′-hydroxy oligonucleotides under reducing condition

We previously reported that reducing-environment-responsive prodrug-type small interfering RNA (siRNA) bearing 2′-O-methyldithiomethyl (2′-O-MDTM) uridine exhibits efficient knockdown activity and nuclease resistance. In this report, we describe the preparation of 2′-O-MDTM oligonucleotides modified not only at uridine but also at adenosine, guanosine and cytidine residues by post-synthetic modification. Precursor oligonucleotides bearing 2′-O-(2,4,6-trimethoxybenzylthiomethyl) (2′-O-TMBTM) adenosine, guanosine, and cytidine were reacted with dimethyl(methylthio)sulfonium tetrafluoroborate to form 2′-O-MDTM oligonucleotides in the same manner as the oligonucleotide bearing 2′-O-TMBTM uridine. Furthermore, the oligonucleotides bearing 2′-O-MDTM adenosine, guanosine, and cytidine were efficiently converted into corresponding natural 2′-hydroxy oligonucleotides under the cytosol-mimetic reducing condition.