Solid-phase RNA Synthesis Research Articles

Chromophoric 5′‐O‐Silyl Protection of N‐Protected 2′‐ACE Ribonucleosides for Solid‐Phase RNA Synthesis

Recent advances in the understanding of the pivotal roles played by endogenous small RNAs in gene regulation have resulted in a substantial and rapidly growing market for synthetic RNA. 5'-Silyl-2'-ACE chemistry has proven to be a robust and reliable technology for the synthesis of oligoribonucleotides. This unit describes an important improvement to this chemistry, by adding a cycle-to-cycle traceability analogous to that inherent in 5'-dimethoxytrityl-based approaches. This is achieved by first regioselectively introducing a 5'-alkynylsilyl protecting group onto the 2'-ACE-protected nucleosides. The 5'-alkynylsilyl group is then reacted with an azide derivative of the chromophore Disperse Red 1, which enables spectrophotometric interrogation of each coupling step following 5'-deprotection. Finally, the protected nucleosides are elaborated into their 3'-phosphoramidite derivatives for use in solid-phase RNA synthesis.

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.

Synthesis of a guanosine phosphoramidite derivative containing a photocleavable protecting group at the O6 position via regioselective protection of the 2'-hydroxy group with a TBDMS group

To construct modified RNA that form A-type duplex with photo-irradiation, a photocleavable a-methyl-2-nitropiperonyl (MeNP) group was introduced at O(6) position of guanosine. A guanosine phosphoramidite derivative containing the MeNP group was synthesized via regioselective 2'-O-protection of 3',5'-O-di(t-butyl)silanediylguanosine with TBDMS group. The MeNP group was found to be stable under conditions of solid-phase synthesis of RNA. The MeNP group was also found to be removable by UV irradiation at wavelength of 365 nm for 10 min.

Assessment of 4-Nitrogenated Benzyloxymethyl Groups for 2‘-Hydroxyl Protection in Solid-Phase RNA Synthesis

The search for a 2'-OH protecting group that would impart ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites led to an assessment of 2'-O-(4-nitrogenated benzyloxy)methyl groups through solid-phase RNA synthesis using phosphoramidites 2a-d, 12a, and 14a. These phosphoramidites exhibited rapid and efficient coupling properties. Particularly noteworthy is the cleavage of the 2'-O-[4-(N-methylamino)benzyloxy]methyl groups in 0.1 M AcOH, which led to U19dT within 15 min at 90 degrees C. [reaction: see text]

Preparation of 2′‐Deoxy‐2′‐Methylseleno‐Modified Phosphoramidites and RNA

The derivatization of nucleic acids with selenium is a useful approach to facilitate phase determination during three-dimensional structural analysis by X-ray crystallography. This unit describes (1) the synthesis and characterization of 2'-deoxy-2'-methylseleno (2'-Se-methyl) nucleosides and their corresponding 3'-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite derivatives, (2) the site-specific incorporation of 2'-Se-methyl ribonucleosides into oligoribonucleotides by chemical RNA solid-phase synthesis, and (3) the enzymatic ligation of Se-containing RNA oligonucleotides to produce a biologically relevant RNA sequence.

Synthesis of RNA Using 2‘-O-DTM Protection

tert-Butyldithiomethyl (DTM), a novel hydroxyl protecting group, cleavable under reductive conditions, was developed and applied for the protection of 2'-OH during solid-phase RNA synthesis. This function is compatible with all standard protecting groups used in oligonucleotide synthesis, and allows for fast and high-yield synthesis of RNA. Oligonucleotides containing the 2'-O-DTM groups can be easily deprotected under the mildest possible aqueous and homogeneous conditions. The preserved 5'-O-DMTr function can be used for high-throughput cartridge RNA purification.

Syntheses of RNAs with up to 100 Nucleotides Containing Site-Specific 2‘-Methylseleno Labels for Use in X-ray Crystallography

The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1,4-dimercapto-2,3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.

Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion.

We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2'-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2'-methylseleno cytidine phosphoramidite and improved the accessibility of the 2'-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2'-selenium derivatization had no effect on the stability of the duplexes with the 3'-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3'-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2'-Se-C and 2'-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2'-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.

The Synthesis of 2?- O -[(Triisopropylsilyl)oxy] methyl ( TOM ) Phosphoramidites of Methylated Ribonucleosides ( m 1 G , m 2 G , m 2 2 G , m 1 I , m 3 U , m 4 C , m 6 A , m 6 2 A ) for Use in Automated RNA Solid-Phase Synthesis

The straightforward synthesis of eight methylated ribonucleoside phosphoramidites is described. These building blocks allow for incorporation of the naturally occuring nucleosides 1-methylguanosine (m1G), N2-methylguanosine (m2G), N2,N2-dimethylguanosine (m22G), 1-methylinosine (m1I), 3-methyluridine (m3U), N4-methylcytidine (m4C), N6-methyladenosine (m6A), and N6,N6-dimethyladenosine (m62A) into oligoribonucleotides by automated RNA solid-phase synthesis. In all cases, the ribose 2′-hydroxyl group of the building blocks is masked by the recently introduced [(triisopropylsilyl)oxy]methyl (TOM) group.

Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin

The synthesis of a 5′- O-BzH-2′- O-ACE-protected-3-methyluridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The phosphoramidite was employed in solid-phase RNA synthesis to generate a series of RNA hairpins containing single or multiple modifications, including the common nucleoside pseudouridine. Three 19-nucleotide hairpin RNAs that represent the 1920-loop region (G 1906–C 1924) of Escherichia coli 23S ribosomal RNA were generated. Modifications were present at positions 1911, 1915, and 1917. The stabilities and structures of the three RNAs were examined by using thermal melting, circular dichroism, and NMR spectroscopy

Chemical Synthesis of 4-Thiouridine-Containing Substrate Analogues of tRNA:Pseudouridine-55 Synthase: Photocross-Linking Studies

For site specific incorporation of 4-thiouridine into oligoribonucleotides a new phosphoramidite is proposed. It makes use of the S-pivaloyloxymethyl group for the protection of the thiol function. This group is easily introduced and removed without modification of the standard protocol for solid phase synthesis of RNA. Three 4-thiouridine-containing oligoribonucleotides (21-mers), corresponding to tRNA minisubstrates of yeast tRNA:pseudouridine-55 synthase (Pus4) were prepared. These 4-thiouridine containing substrates were characterized and used as photoaffinity probe of the enzyme:substrate complex. Irradiation resulted in the specific photocross-linking of these oligoribonucleotides with purified recombinant tRNA:pseudouridine-55 synthase.

1, 1, 3, 3-Tetraisopropyl-3-(2-(triphenylmethoxy)ethoxy)disiloxane-1-yl group, a potential 5′-O-protecting group for solid-phase RNA synthesis

Abstract 1, 1, 3, 3-Tetraisopropyl-3-(2-(triphenylmethoxy)ethoxy)disiloxane-1-yl (TES) group was designed for solid-phase RNA synthesis as a 5′-O-protecting group, which can be combined with the tetrahydropyranyl group, a 2′-hydroxyl protecting group. The TES group can be selectively introduced to the 5′-hydroxy group of nucleosides and can be easily removed by 0.1M tetrabutylammonium fluoride or 0.01M hydrochloric acid.

The synthesis of allyl- and allyloxycarbonyl-protected RNA phosphoramidites. Useful reagents for solid-phase synthesis of RNAs with base-labile modifications

Abstract The synthesis of allyl- and allyloxycarbonyl (AOC)-protected RNA phosphoramidites is reported. The use of allyl and AOC groups allows the chemistry of solid-phase RNA synthesis to be expanded to include base-labile modified nucleosides, such as acetylcytidine. The allyl- and AOC-protective chemistry can be further expanded for the construction of RNAs on solid supports and affinity columns.

A Chemical Method for Site-Specific Modification of RNA: The Convertible Nucleoside Approach

Knowledge of RNA structure can greatly facilitate the understanding of its biological function. However, the physical properties of RNA, especially its conformational heterogeneity, present an impediment to high-resolution structural analysis. Thus, lower resolution methods such as biochemical probing, phylogenetic analysis, and molecular modeling have come to serve an important role in RNA science. This situation has created the need for a means by which to constrain RNA structure, either to reduce its conformational flexibility or to help distinguish between alternative structural models. To address this need, we have developed chemistry that permits the site-specific introduction of functionalizable tethers into RNA. Here we report the design and synthesis of reagents for use in solid-phase RNA synthesis that allow the functionalization of the base moiety of G, C, and A residues. Upon incorporation into oligonucleotides and subsequent treatment with alkylamines, the convertible nucleoside derivatives r...

Improvements to the Chemical Synthesis of Biologically Active RNA Using 2'-O-Fpmp Chemistry

Abstract The synthetic cycle protocol for the solid phase synthesis of RNA using 5′-O-(DMTr)-2′-O-(Fpmp)-ribonucleoside phosphoramidites is optimised. A simple and reliable two step deprotection procedure is developed to isolate biologically active RNA. It is demonstrated that fully deprotected RNA is completely stable under the deprotection conditions and that it does not undergo internucleotide cleavage and/or migration. Ribozymes and substrate RNAs synthesized using this chemistry were found to be catalytically active.

Rapid synthesis of oligoribonucleotides using 2′- O-( o-nitrobenzyloxymethyl)-protected monomers

A new, easily introduced protecting group for ribonucleoside 2′-hydroxyls, o-nitrobenzyloxymethyl, permits fast, effective solid phase synthesis of RNA.

New nucleoside phosphoramidites and coupling protocols for solid-phase RNA synthesis

The 5'-O-(4,4'-dimethoxytrityl)-2'-O-(trialkylsilyl) ribonucleoside 3'-O-(2-cyanoethyl N,N-diethylphosphoramidites) modified monomers for RNA synthesis, were prepared from 2-cyanoethyl N,N-diethylchlorophosphoramidite. In conjunction with newly developed coupling protocols for automated solid-phase synthesis, they afforded synthetic oligoribonucleotides up to 74 base units in length. Purification procedures by reverse phase HPLC and PAGE methods are also presented

Selective 2′-benzoylation at the 2′, 3′-diols of protected ribonucleosides. New solid phase synthesis of RNA and DNA-RNA mixtures

5'-0-(Dimethoxytrityl)-2'-0-(benzoyl or 3,4,5-trimethoxybenzoyl)-base protected ribonucleosides have been prepared by selective benzoylation of the 2'-hydroxyl group. The isomerization of the 2'-benzoates to the 3'-benzoates was studied. The protected ribonucleosides have been converted to either methylphosphochloridites or methylphosphoamidites and used to synthesize oligoribonucleotides on silica gel solid support. The synthetic RNA were deprotected and isolated using conditions that minimize internucleotide cleavage. The use of 2'-benzoates as protecting groups for ribonucleosides has made it possible to easily prepare and isolate mixtures of DNA and RNA.