Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2′-O-Methyl (2′OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2′OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2′OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application.     

Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway.     

Synthesis, cellular uptake and HIV-1 Tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides

Oligonucleotides composed of 2′-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5′-disulphide-linked conjugates of 3′-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48–58), Penetratin and R₉F₂ was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48–58), Penetratin and R₉ conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R₉F₂ and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.     

Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5′-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.     

Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8–15 nucleotides and by the incorporation of high affinity RNA binders such as 2′, 4′-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2′,4′-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4–5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.     

Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.     

m6A methylation potentiates cytosolic dsDNA recognition in a sequence-specific manner

Nucleic acid sensing through pattern recognition receptors is critical for immune recognition of microbial infections. Microbial DNA is frequently methylated at the N⁶ position of adenines (m6A), a modification that is rare in mammalian host DNA. We show here how that m6A methylation of 5′-GATC-3′ motifs augments the immunogenicity of synthetic double-stranded (ds)DNA in murine macrophages and dendritic cells. Transfection with m6A-methylated DNA increased the expression of the activation markers CD69 and CD86, and of Ifnβ, iNos and Cxcl10 mRNA. Similar to unmethylated cytosolic dsDNA, recognition of m6A DNA occurs independently of TLR and RIG-I signalling, but requires the two key mediators of cytosolic DNA sensing, STING and cGAS. Intriguingly, the response to m6A DNA is sequence-specific. m6A is immunostimulatory in some motifs, but immunosuppressive in others, a feature that is conserved between mouse and human macrophages. In conclusion, epigenetic alterations of DNA depend on the context of the sequence and are differentially perceived by innate cells, a feature that could potentially be used for the design of immune-modulating therapeutics.     

Triplex formation with 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) having C3'-endo conformation at physiological pH

Antigenes, which are substances that inhibit gene expression by binding to double-stranded DNA (dsDNA) in a sequence-specific manner, are currently sought for the treatment of various gene-related diseases. As such antigenes, we developed new nuclease-resistant oligopyrimidine nucleotides that are partially modified with 2′-O,4′-C-ethylene nucleic acids (ENA), which are constrained in the C3′-endo conformation and can form a triplex with dsDNA at physiological pH. It was found that these oligonucleotides formed triplexes similarly to those partially modified with 2′-O,4′-C-methylene nucleic acids (2′,4′-BNA or LNA), as determined by UV melting analyses, electromobility shift assays, CD spectral analyses and restriction enzyme inhibition assays. In our studies, oligonucleotides fully modified with ENA have δ torsion angle values that are marginally higher than those of 2′,4′-BNA/LNA. ENA oligonucleotides present in 10-fold the amount of dsDNA were found to be favorable in forming triplexes. These results provide useful information for the future design of triplex-forming oligonucleotides fully modified with such nucleic acids constrained in the C3′-endo conformation considering that oligonucleotides fully modified with 2′,4′-BNA/LNA do not form triplexes.     

RNA cleavage products generated by antisense oligonucleotides and siRNAs are processed by the RNA surveillance machinery

DNA-based antisense oligonucleotides (ASOs) elicit cleavage of the targeted RNA by the endoribonuclease RNase H1, whereas siRNAs mediate cleavage through the RNAi pathway. To determine the fates of the cleaved RNA in cells, we lowered the levels of the factors involved in RNA surveillance prior to treating cells with ASOs or siRNA and analyzed cleavage products by RACE. The cytoplasmic 5′ to 3′ exoribonuclease XRN1 was responsible for the degradation of the downstream cleavage products generated by ASOs or siRNA targeting mRNAs. In contrast, downstream cleavage products generated by ASOs targeting nuclear long non-coding RNA Malat 1 and pre-mRNA were degraded by nuclear XRN2. The downstream cleavage products did not appear to be degraded in the 3′ to 5′ direction as the majority of these products contained intact poly(A) tails and were bound by the poly(A) binding protein. The upstream cleavage products of Malat1 were degraded in the 3′ to 5′ direction by the exosome complex containing the nuclear exoribonuclease Dis3. The exosome complex containing Dis3 or cytoplasmic Dis3L1 degraded mRNA upstream cleavage products, which were not bound by the 5′-cap binding complex and, consequently, were susceptible to degradation in the 5′ to 3′ direction by the XRN exoribonucleases.     

Infection-induced 5′-half molecules of tRNAHisGUG activate Toll-like receptor 7

Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5′-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5′-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5′-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5′-tRNA^HisGUG half molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5′-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5′-tRNA half species into EVs. The EV-5′-tRNA^HisGUG half was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5′-tRNA half molecules was also observed in the plasma of patients infected with Mycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of “immune activators” to 5′-tRNA half molecules.

Although Toll-like receptors (TLRs) play a crucial role in the innate immune response, their endogenous ligands have not been fully explored. This study identifies tRNA half-molecules as abundant TLR ligands which are upregulated upon infection by mycobacteria and activate TLR7.     

Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5′ppp- mRNAs

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K_1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K_1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K_1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.     

Building unique bonds to fight misplaced DNA

A cyclic dinucleotide comprised of GMP and AMP was previously shown to be a key intermediate during activation of innate immune responses to cytosolic DNA. A report by Patel and Tuschl groups published in Cell reveals the structure of the enzyme involved in the synthesis of this second messenger and identifies this cyclic dinucleotide as a unique compound in metazoan cell signaling.For more than 100 years it has been known that DNA stimulates immune responses¹. Hence, when DNA reaches the cytoplasmic compartment in a cell, no matter originating from an infectious agent like viruses or from the damaged nucleus or mitochondria, it is recognized as a sign of danger. DNA can provoke severe consequences as it can be seen from aberrant recognition of lost DNA in autoimmune conditions such as systemic lupus erythematous and Sjogren''s syndrome. To perceive such a dreadful insult, several DNA-sensing proteins are present in mammalian cells. Some of these DNA sensors activate a cytoplasmic protein called stimulator of interferon (IFN) genes (STING). STING then turns on a series of protein kinases, culminating in the production of type I IFNs and other cytokines that participate in host immune responses². Gaining details about the structures and the mechanisms associated with such cellular responses has been a matter of great interest in the immunology field and may bear relevance for both infectious and autoimmune conditions.It was recently demonstrated that STING activation by DNA is mediated by a cyclic dinucleotide comprised of GMP and AMP, called cGAMP. Hence, upon infection with DNA viruses or delivery of DNA into the cytoplasm of some immune cells, cGAMP levels build up, and the dinucleotide binds directly to STING, leading to type I IFN production through activation of IRF3 via TBK1³. Therefore, cGAMP acts as a second messenger during DNA-triggered innate immune response. It was also shown that cGAMP synthesis relies on the activity of the enzyme cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family⁴. cGAS, therefore, acts as a cytoplasmic DNA sensor that generates the second messenger cGAMP, essential for activating STING-mediated type I IFN production.Cyclic dinucleotides are well-known bacterial intracellular signal transducers, and cyclic di-GMP (c-di-GMP) has been acknowledged as a universal bacterial second messenger⁵. The structural and biochemical analysis of the bacterial enzymes responsible for the synthesis of this second messenger suggested that c-di-GMP is formed from two molecules of GTP via a two-step reaction that generates a 3′-5′-phosphodiester linkage between the two GMP nucleotides⁶. Taking the bacterial synthesis as a model and based on the fact that chemically synthesized cGAMP with the 3′-5′-phosphodiester linkage stimulates STING-dependent type I IFN production in mammalian cells³, one would assume that cGAS-derived cGAMP likely contains the same phosphodiester linkage. However, in an outstanding paper published by Cell, Gao et al.⁷ challenged this view. Combining structural, chemical, biochemical and biological techniques, they definitely establish that cGAMP contains a 2′-5′ linkage, position this second messenger as the first 2′-5′ linkage-containing metazoan second messenger ever described, and distinguish it from the bacterial cyclic dinucleotides. The previous study had concluded that the form of cGAMP generated in mammalian cells was a 3′-5′-phosphodiester nucleotide. In this study, however, Gao et al. identify cGAMP as actually cyclic [G(2′,5′)pA(3′,5′)p] cGAMP. This form is unique to metazoans. The bacterial form is therefore subtly different and is less potent as an activator of STING³.As a first approach for understanding the mechanisms involved in cGAMP synthesis after DNA recognition, the authors compared the structure of the crystalized cGAS in its free state with the structure of the enzyme complexed with double-stranded DNA (dsDNA). dsDNA interaction with the enzyme led to pronounced conformational changes on the protein, allowing cGAS to adopt a catalytically competent conformation, a feature considered to be essential for a cytosolic DNA sensor. Comparison of the structures of the dsDNA-bound cGAS complexed with GTP, or with GMP + ATP or with GTP + ATP suggested that one of the phosphodiester linkages in the dinucleotide produced by the reaction was of the 2′-5′ nature, in contrast to the previously assumed 3′-5′ conformation. This unexpected result was supported by biochemical analysis and confirmed after comparison of the purified cGAS-derived product with chemically synthesized dinucleotide standards.The authors have also provided evidence suggesting that cyclization occurs in a stepwise manner and showed that a pair of divalent cations is necessary for phosphodiester bond formation. Finally, the use of functional mutants of the dsDNA-binding site or of the catalytic pocket of cGAS reinforced the conclusions gained from the structural analysis, confirming the importance of complex formation between cGAS and dsDNA and of the nucleotide-interacting residues in the catalytic pocket for activity of cGAS and consequent STING-mediated type I IFN production.The great amount of data presented by Gao et al. provide detailed information regarding the synthesis of cGAMP by cGAS, and valuable knowledge for understanding the control of cellular responses to cytosolic DNA (Figure 1). Although 2′-5′ bonds were previously shown to occur in mammalian biochemical reactions during the polymerization of ATP into linear oligoadenilate by the dsRNA sensor oligoadenylate synthetase 1 (OAS1)⁸, this is the first documented case of such a linkage in dinucleotides. This kind of phosphodiester bond is uncommon, and few nucleases are reported to be able to hydrolyze 2′-5′ linkages⁹. This might promote a greater stability of the second messenger in cells and consequently enable effective and, maybe, long-lasting signal transduction. This unique structure establishes cGAMP as a founding member of a potentially broader class of metazoan second messengers. Importantly, the fact that the second messenger and the enzymes involved in dinucleotide synthesis in bacterial systems present some structural distinctions from the ones found in metazoan cells points to features that may be explored for selectively targeting the prokaryotic or the metazoan pathway. In addition, although it was not clearly demonstrated that cGAS and cGAMP directly impact in autoimmune responses, the structural and biochemical information provided by Gao et al. may bear relevance for the development of small molecule inhibitors with therapeutic potentials in such conditions.Open in a separate windowFigure 1The study by Gao et al. shows that, upon DNA recognition in cytoplasm, cGAS suffers a conformational shift that allows it to convert GTP and ATP nucleotides into the cyclic compound cGAMP, containing the [G(2′,5′)pA(3′,5′)p] linkages. The 2′-5′ linkage is a unique feature of metazoan cyclic dinucleotides, as bacterial ones described so far present exclusively 3′-5′ phosphodiester bonds. cGAMP subsequently binds to STING, leading to TBK1-mediated IRF3 activation and robust type I IFN production.     

Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals

A series of antisense oligonucleotides (ASOs) containing either 2′-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights. This toxicity was evident for multiple sequences targeting three different biological targets, as well as in mismatch control sequences having no known mRNA targets. Histopathological evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was observed as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.     

Structure-function correlations derived from faster variants of a RNA ligase deoxyribozyme

We previously reported the in vitro selection of several Mg²⁺-dependent deoxyribozymes (DNA enzymes) that synthesize a 2′–5′ RNA linkage from a 2′,3′-cyclic phosphate and a 5′-hydroxyl. Here we subjected the 9A2 deoxyribozyme to re-selection for improved ligation rate. We found two new DNA enzymes (7Z81 and 7Z48) that contain the catalytic core of 7Q10, a previously reported small deoxyribozyme that is unrelated in sequence to 9A2. A third new DNA enzyme (7Z101) is unrelated to either 7Q10 or 9A2. The new 7Z81 and 7Z48 DNA enzymes have ligation rates over an order of magnitude higher than that of 7Q10 itself and they have additional sequence elements that correlate with these faster rates. Our findings provide insight into structure–function relationships of catalytic nucleic acids.     

A Point Mutation in the Hair Cell Nicotinic Cholinergic Receptor Prolongs Cochlear Inhibition and Enhances Noise Protection

The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9′T line of knockin mice with a threonine for leucine change (L9′T) at position 9′ of the second transmembrane domain of the α9 nicotinic cholinergic subunit, rendering α9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9′T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9′T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the α9α10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9^{L9′T/L9′T} mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter α9α10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.     

2'-Methylseleno-modified oligoribonucleotides for X-ray crystallography synthesized by the ACE RNA solid-phase approach

Site-specifically modified 2′-methylseleno RNA represents a valuable derivative for phasing of X-ray crystallographic data. Several successful applications in three-dimensional structure determination of nucleic acids, such as the Diels–Alder ribozyme, have relied on this modification. Here, we introduce synthetic routes to 2′-methylseleno phosphoramidite building blocks of all four standard nucleosides, adenosine, cytidine, guanosine and uridine, that are tailored for 2′-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis. We additionally report on their incorporation into oligoribonucleotides including deprotection and purification. The methodological expansion of 2′-methylseleno labeling via ACE RNA chemistry is a major step to make Se-RNA generally accessible and to receive broad dissemination of the Se-approach for crystallographic studies on RNA. Thus far, preparation of 2′-methylseleno-modified oligoribonucleotides has been restricted to the 2′-O-[(triisopropylsilyl)oxy]methyl (TOM) and 2′-O-tert-butyldimethylsilyl (TBDMS) RNA synthesis methods.