O-methyl Oligoribonucleotides Research Articles

2'-O-Methyl Oligoribonucleotide Analogs Used to Change the Temperature Characteristics of Immobilized Probes and to Enhance the Specificity of Hybridization

The specificity of interactions between oligonucleotide probes immobilized in a biological microchip (biochip) and target DNA depends on a number of factors, with the main roles being played by the probe length and nucleotide sequence (GC composition primarily). The 2'-O-methyl oligoribonucleotides are known to form more stable duplexes with complementary DNA compared with 2'-deoxyribonucleotide probes. The study tested the possibility of using 2'-O-methyl oligoribonucleotides to increase the specificity of interactions between target DNA and probes immobilized in a biochip. Fluorophore-labeled target DNA was obtained via one-round asymmetric PCR with simultaneous incorporation of a fluorescent label in the PCR product. After hybridization with immobilized probes, fluorescent signals from gel pads with 2'-O-methyl oligoribonucleotide probes were, on average, two times higher than from those with 2'-deoxyribonucleotide probes, with the target DNA sequences and probe concentrations being the same. The increase in fluorescence intensity was greater in the case of perfect versus imperfect (mismatched) duplexes.

Interactions of 2'-O-methyl oligoribonucleotides with the RNA models of the 30S subunit A-site.

Synthetic oligonucleotides targeting functional regions of the prokaryotic rRNA could be promising antimicrobial agents. Indeed, such oligonucleotides were proven to inhibit bacterial growth. 2’-O-methylated (2’-O-Me) oligoribonucleotides with a sequence complementary to the decoding site in 16S rRNA were reported as inhibitors of bacterial translation. However, the binding mode and structures of the formed complexes, as well as the level of selectivity of the oligonucleotides between the prokaryotic and eukaryotic target, were not determined. We have analyzed three 2’-O-Me oligoribonucleotides designed to hybridize with the models of the prokaryotic rRNA containing two neighboring aminoglycoside binding pockets. One pocket is the paromomycin/kanamycin binding site corresponding to the decoding site in the small ribosomal subunit and the other one is the close-by hygromycin B binding site whose dynamics has not been previously reported. Molecular dynamics (MD) simulations, as well as isothermal titration calorimetry, gel electrophoresis and spectroscopic studies have shown that the eukaryotic rRNA model is less conformationally stable (in terms of hydrogen bonds and stacking interactions) than the corresponding prokaryotic one. In MD simulations of the eukaryotic construct, the nucleotide U1498, which plays an important role in correct positioning of mRNA during translation, is flexible and spontaneously flips out into the solvent. In solution studies, the 2’-O-Me oligoribonucleotides did not interact with the double stranded rRNA models but all formed stable complexes with the single-stranded prokaryotic target. 2’-O-Me oligoribonucleotides with one and two mismatches bound less tightly to the eukaryotic target. This shows that at least three mismatches between the 2’-O-Me oligoribonucleotide and eukaryotic rRNA are required to ensure target selectivity. The results also suggest that, in the ribosome environment, the strand invasion is the preferred binding mode of 2’-O-Me oligoribonucleotides targeting the aminoglycoside binding sites in 16S rRNA.

Restoration of correct splicing in IVSI-110 mutation of β-globin gene with antisense oligonucleotides: implications and applications in functional assay development.

Objective(s):The use of antisense oligonucleotides (AOs) to restore normal splicing by blocking the recognition of aberrant splice sites by the spliceosome represents an innovative means of potentially controlling certain inherited disorders affected by aberrant splicing. Selection of the appropriate target site is essential in the success of an AO therapy. In this study, in search for a splice model system to facilitate the evaluation of AOs to redirect defective splicing of IVSI-110 β-globin intron, an EGFP-based IVSI-110 specific cellular reporter assay system has been developed and a number of AOs were tested in this cellular splicing assay.Materials and Methods:A recombinant plasmid (pEGFP/I-110) carrying the EGFP gene interrupted by a mutated human β-globin intron 1 (IVSI-110) was developed and transfected into K562 cells. A number of AOs with a 2’-O-methyl oligoribonucleotide (2’-O-Me) backbone system were systematically tested in this cellular splicing assay.Results:The mutation in the intron causes aberrant splicing of EGFP pre-mRNA, preventing translation of EGFP; however, treatment of the cells with specific concentration of a sequence specific 2’-O-Me AO targeted to the aberrant splice site induced correct splicing and resulted in restoring of EGFP activity.Conclusion:This cellular splicing assay provides a novel functional assay system in assessing the cellular delivery efficiency of AOs and therapeutic effect of AOs in restoration of aberrant splicing.

Synthesis of Aminoglycoside-2'-O-Methyl Oligoribonucleotide Fusions.

Phosphoramidite building blocks of ribostamycin (3 and 4), that may be incorporated at any position of the oligonucleotide sequence, were synthesized. The building blocks, together with a previously described neomycin-modified solid support, were applied for the preparation of aminoglycoside-2′-O-methyl oligoribonucleotide fusions. The fusions were used to clamp a single strand DNA sequence (a purine-rich strand of c-Myc promoter 1) to form triple helical 2′-O-methyl RNA/DNA-hybrid constructs. The potential of the aminoglycoside moieties to stabilize the triple helical constructs were studied by UV-melting profile analysis.

Analysis of ribosomal inter-subunit sites as targets for complementary oligonucleotides.

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined. Based on these properties we selected rRNA targets for hybridization with complementary 2'-O-methyl oligoribonucleotides (2'-OMe RNAs). Further, the inhibition efficiencies of the designed ribosome-interfering 2'-OMe RNAs were tested using a β-galactosidase assay in a translation system based on the E. coli extract. Several of the oligonucleotides displayed IC50 values below 1 μM, which were in a similar range as those determined for known ribosome inhibitors, tetracycline and pactamycin. The calculated opening and van der Waals stacking energies of the rRNA targets correlated best with the inhibitory efficiencies of 2'-OMe RNAs. Moreover, the binding affinities of several oligonucleotides to both 70S ribosomes and isolated 30S and 50S subunits were measured using a double-filter retention assay. Further, we applied heat-shock chemical transformation to introduce 2'-OMe RNAs to E. coli cells and verify inhibition of bacterial growth. We observed high correlation between IC50 in the cell-free extract and bacterial growth inhibition. Overall, the results suggest that the computational analysis of potential rRNA targets within the conformationally dynamic regions of inter-subunit bridges can help design efficient antisense oligomers to probe the ribosome function.

In Vivo Bone-Targeting of Bis(phosphonate)-Conjugated Double Helical RNA Monitored by Positron Emission Tomography.

A bis(phosphonate) conjugate of 2'-O-methyl oligoribonucleotide (microRNA-21) was synthesized and used as a bone-targeting carrier in the systemic delivery of a (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated 2'-O-methyl oligoribonucleotide (anti-microRNA-21). The whole-body biodistribution of the double helical RNA was monitored by positron emission tomography (PET), which verified the expected bis(phosphonate)-induced bone accumulation in healthy rats.

Effect of 2'-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells.

Optimizing oligonucleotides as therapeutics will require exploring how chemistry can be used to enhance their effects inside cells. To achieve this goal it will be necessary to fully explore chemical space around the native DNA/RNA framework to define the potential of diverse chemical modifications. In this report we examine the potential of thiophosphonoacetate (thioPACE)-modified 2'-O-methyl oligoribonucleotides as inhibitors of human huntingtin (HTT) expression. Inhibition occurred, but was less than with analogous locked nucleic acid (LNA)-substituted oligomers lacking the thioPACE modification. These data suggest that thioPACE oligonucleotides have the potential to control gene expression inside cells. However, advantages relative to other modifications were not demonstrated. Additional modifications are likely to be necessary to fully explore any potential advantages of thioPACE substitutions.

Synthesis of multi-galactose-conjugated 2′-O-methyl oligoribonucleotides and their in vivo imaging with positron emission tomography

68Ga labelled 2′-O-methyl oligoribonucleotides (anti-miR-15b) bearing one, three or seven d-galactopyranoside residues have been prepared and their distribution in healthy rats has been studied by positron emission tomography (PET). To obtain the heptavalent conjugate, an appropriately protected 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) precursor bearing a 4-[4-(4,4′-dimethoxytrityloxy)butoxy]phenyl side arm was first immobilized via a base labile linker to the support and the oligonucleotide was assembled on the detritylated hydroxyl function of this handle. A phosphoramidite building block bearing two phthaloyl protected aminooxy groups and one protected hydroxyl function was introduced into the 5′-terminus. One acetylated galactopyranoside was coupled as a phosphoramidite to the hydroxyl function, the phthaloyl protections were removed on-support and two trivalent galactopyranoside clusters were attached as aldehydes by on-support oximation. A two-step cleavage with aqueous alkali and ammonia released the conjugate in a fully deprotected form, allowing radiolabelling with 68Ga in solution. The mono- and tri-galactose conjugates were obtained in a closely related manner. In vivo imaging in rats with PET showed remarkable galactose-dependent liver targeting of the conjugates.

Synthesis and properties of oligonucleotides modified with 2'-O-(2-carboxyethyl)nucleotides and their carbamoyl derivatives.

2'-O-Methyl oligoribonucleotides with four kinds of 2'-O-modified uridine derivatives were synthesised. Their duplex stability, hydration behavior and exonuclease resistance were studied by spectroscopic analyses and molecular dynamics simulations. Consequently, 2'-O-modification of the uridine residue with 2-carbamoylethyl or 2-(N-methylcarbamoyl)ethyl groups resulted in a significant improvement of the exonuclease resistance without the loss of duplex stability.

4′-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine as a Sensitive 19F NMR Sensor for the Detection of Oligonucleotide Secondary Structures

4'-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine was synthesized and incorporated as a phosphoramidite into oligonucleotide sequences. Its applicability as a sensor for the (19)F NMR spectroscopic detection of DNA and RNA secondary structures was demonstrated. On DNA, the (19)F NMR measurements were focused on monitoring of duplex-triplex conversion, for which this fluorine-labeled 2'-deoxynucleoside proved to be a powerful sensor. This sensor seemingly favors DNA, but its behavior in the RNA environment also turned out to be informative. As a demonstration, invasion of a 2'-O-methyl oligoribonucleotide into an RNA hairpin model (HIV-1 TAR) was monitored by (19)F NMR spectroscopy. According to the thermal denaturation studies by UV spectroscopy, the effect of the 4'-C-(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl moiety on the stability of these DNA and RNA models was marginal.

2,6-Bis(functionalized) purines as metal-ion-binding surrogate nucleobases that enhance hybridization with unmodified 2′-O-methyl oligoribonucleotides

The capacity of three different purine bases, viz. 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, 2-(3,5-dimethylpyrazol-1-yl)adenine and 2,6-bis(2-acetyl-1-methylhydrazino)purine, to form metal-ion mediated base pairs with the native nucleobases has been examined. For this purpose, ribonucleosides derived from these bases were incorporated into an intrastrand or a 3'-terminal position of short 2'-O-methyl oligoribonucleotides and the hybridization properties of these base modified oligomers in the absence and presence of three different metal ions (Cu(2+), Zn(2+) and Pd(2+)) were studied by UV- and CD-spectrometry. The first two bases were found to stabilize short oligonucleotide duplexes when incorporated into the 3'-termini of both strands, even in the absence of divalent metal ions but especially in the presence of Cu(2+). The highest melting temperature determined for such a duplex was 71.8 °C, nearly 30 °C higher than the T(m) of the respective solely Watson-Crick paired duplex. Despite the dramatic stabilizing effect of the terminal metallo-base pairs, these short modified oligonucleotides retained sequence-selectivity for the internal Watson-Crick base pairs: two internal mismatches dropped the melting temperature to 10-11 °C. In an internal position, only 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, which in the absence of metal ions was destabilizing, exhibited metal-ion-dependent stabilization of duplex formation with unmodified 2'-O-methyl oligoribonucleotides. The melting temperature in the presence of Cu(2+) was increased from 6 to 14 °C, depending on the identity of the opposite base.

Synthesis and biological activity of phosphonoacetate- and thiophosphonoacetate-modified 2′-O-methyl oligoribonucleotides

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.

Metal Ion Chelates as Surrogates of Nucleobases for the Recognition of Nucleic Acid Sequences: ThePd2+Complex of 2,6-Bis(3,5-dimethylpyrazol-1-yl)purine Riboside

A 2,6-bis(3,5-dimethylpyrazol-1-yl)purine ribonucleoside has been prepared and incorporated as a conventionally protected phosphoramidite into a 9-mer 2′-O-methyl oligoribonucleotide. According to 1H NMR spectroscopic studies, this nucleoside forms with Pd2+ and uridine a ternary complex that is stable at a micromolar concentration range. CD spectroscopic studies on oligonucleotide hybridization, in turn, suggest that the Pd2+ chelate of this artificial nucleoside, when incorporated in a 2′-O-methyl-RNA oligomer, is able to recognize thymine within an otherwise complementary DNA strand. The duplex containing thymidine opposite to the artificial nucleoside turned out to be somewhat more resistant to heating than its counterpart containing 2′-deoxycytidine in place of thymidine, but only in the presence of Pd2+. According to UV-melting measurements, replacement of 2′-O-methyladenosine with the artificial nucleoside markedly enhances hybridization with a DNA target, irrespective of the identity of the opposite base and the presence of Pd2+. With the thymidine containing DNA target, the T m value is 2–4°C higher than with targets containing any other nucleoside opposite to the artificial nucleoside, but the dependence on Pd2+ is much less clear than in the case of the CD studies.

Synthesis of Aminoglycoside-3′-Conjugates of 2′-O-Methyl Oligoribonucleotides and Their Invasion to a 19F labeled HIV-1 TAR Model

The potential of aminoglycosides to induce RNA-invasion has been demonstrated. For this purpose, aminoglycoside-3'-conjugates of 2'-O-methyl oligoribonucleotides have been synthesized entirely on a solid phase. The synthesis includes an automated oligonucleotide chain elongation to solid-supported neomycin, ribostamycin, and methyl neobiosamine, and a two-step deprotection/release of the solid-supported conjugate, which allows exploitation of a simple protecting group scheme. Conjugates have been targeted to a (19)F labeled HIV-1 TAR RNA model (Trans Activation Response element of HIV), which allows monitoring of the invasion by (19)F NMR spectroscopy. A remarkably enhanced invasion, compared to that resulting from the corresponding unmodified 2'-O-methyl oligoribonucleotide (5'-CAGGCUCA-3'), has been obtained by the neomycin conjugate. The increased affinity results from a cooperative binding of the neomycin moiety and hybridization, though the invasion may also follow a mechanism, in which the first molar equivalent of the conjugate induces hybridization of the second.

The Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane as an Artificial Nucleobase

{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4′-dimethoxytrityl)-α-D- erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1- yl]ethyl}acetamide (1) was prepared and incorporated into a 2′-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2′-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn2+. Introduction of Zn2+ moderately stabilized the duplex with U-bulged targets.

A growing molecular toolbox for the functional analysis of microRNAs in Caenorhabditis elegans.

With the growing number of microRNAs (miRNAs) being identified each year, more innovative molecular tools are required to efficiently characterize these small RNAs in living animal systems. Caenorhabditis elegans is a powerful model to study how miRNAs regulate gene expression and control diverse biological processes during development and in the adult. Genetic strategies such as large-scale miRNA deletion studies in nematodes have been used with limited success since the majority of miRNA genes do not exhibit phenotypes when individually mutated. Recent work has indicated that miRNAs function in complex regulatory networks with other small RNAs and protein-coding genes, and therefore the challenge will be to uncover these functional redundancies. The use of miRNA inhibitors such as synthetic antisense 2'-O-methyl oligoribonucleotides is emerging as a promising in vivo approach to dissect out the intricacies of miRNA regulation.

Solid-Supported 2′-O-Glycoconjugation of Oligonucleotides by Azidation and Click Reactions

2'-O-[(2-Bromoethoxy)methyl]cytidine and 2'-O-[(2-azidoethoxy)methyl]cytidine have been prepared and introduced as appropriately protected 3'-phosphoramidite (1) and 3'-(H-phosphonate) (2) building blocks, respectively, into 2'-O-methyl oligoribonucleotides. The support-bound oligonucleotides were subjected to two consecutive conjugations with alkynyl-functionalized monosaccharides. The first saccharide was introduced by a Cu(I) promoted click reaction with 2 and the second by azidation of the 2-bromoethoxy group of 1 followed by the click reaction. The influence of the 2'-glycoconjugations on hybridization with DNA and 2'-O-methyl RNA targets was studied. Two saccharide units within a 15-mer oligonucleotide had a barely noticeable effect on the duplex stability, while introduction of a third one moderately decreased the melting temperature.

Characterization of RNA Invasion by 19F NMR Spectroscopy

(19)F NMR spectroscopy offers an efficient tool for monitoring RNA invasion. The invasion of 2'-O-methyl oligoribonucleotides into a (19)F-labeled HIV-1 TAR RNA model and the temperature-dependent behavior of the complexes obtained have been examined.

Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent

BackgroundAntisense reagents can serve as efficient and versatile tools for studying gene function by inhibiting nucleic acids in vivo. Antisense reagents have particular utility for the experimental manipulation of the activity of microRNAs (miRNAs), which are involved in the regulation of diverse developmental and physiological pathways in animals. Even in traditional genetic systems, such as the nematode Caenorhabditis elegans, antisense reagents can provide experimental strategies complementary to mutational approaches. Presently no antisense reagents are available for inhibiting miRNAs in the nematode C. elegans.ResultsWe have developed a new class of fluorescently labelled antisense reagents to inhibit miRNAs in developing worms. These reagents were synthesized by conjugating dextran with 2'-O-methyl oligoribonucleotide. The dextran-conjugated antisense reagents can be conveniently introduced into the germline of adult hermaphrodites and are transmitted to their progeny, where they efficiently and specifically inhibit a targeted miRNA in different tissues, including the hypodermis, the vulva and the nervous system. We show that these reagents can be used combinatorially to inhibit more than one miRNA in the same animal.ConclusionThis class of antisense reagents represents a new addition to the toolkit for studying miRNA in C. elegans. Combined with numerous mutants or reporter stains available, these reagents should provide a convenient approach to examine genetic interactions that involve miRNA, and may facilitate studying functions of miRNAs, especially ones whose deletion strains are difficult to generate.See related research article: http://jbiol.com/content/9/3/20

Development of peptide-oligonucleotide conjugates for regulation of small RNA function

Recently, various microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified and play important roles in gene regulatory networks. However it is a little known about their biological functions. These small RNAs exhibit their function to form a ribonucleoprotein complex, the RISC (RNA-induced silencing complex), which modulates gene expression by translational repression. In this study, we developed a novel peptide antagonist to inhibit the RISC function. The peptide was conjugated to 2'-O-methyl oligoribonucleotides, which have a complementary sequence to the guide strand of siRNA, and regulatory effects of the peptide on RISC activity were examined. It was revealed that the peptide drastically enhanced inhibitory effects of the oligonucleotide on RISC activity. Here we demonstrate our peptide-oligonucleotide conjugate that can provide a powerful and specific way to regulate the small RNA function.