Molecular design of a eukaryotic messenger RNA and its chemical synthesis

Iwase, Reiko; Maeda, Masayuki; Fujiwara, Toshiki; Sekine, Mitsuo; Hata, Tsujiaki; Miura, Kiyonori

doi:10.1093/nar/20.7.1643

Cited by 22 publications

(6 citation statements)

References 47 publications

(47 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Alternatively, capped RNA can be produced by using purely chemical synthesis, as has been reported for m 3 2,2,7 GpppAmpUmpA and m 7 GpppGUAUUAAUA . Although these methods are efficient, they are more difficult to perform, and the large-scale production of the isotopically labeled molecules required for NMR spectroscopy by such methods is costly.…”

Section: Discussionmentioning

confidence: 99%

Efficient Synthesis of ¹³C,¹⁵N-Labeled RNA Containing the Cap Structure m⁷GpppA

et al. 2000

Self Cite

View full text Add to dashboard Cite

Studying the mechanism by which the cap structure performs its numerous biological roles in mRNA metabolism by NMR spectroscopy requires the large-scale production of isotopically labeled capped RNA. We present an efficient method for production of short RNA containing the cap structure m7GpppA, by combining chemical synthesis with enzymatic synthesis using a DNA primase. This method was employed to synthesize three capped RNA molecules, m7Gppp[13C,15N]A, m7Gppp[13C,15N]ACC, and m7GpppA[13C,15N]C[13C,15N]C, that contain selective 13C,15N-labeled adenosine or cytosine nucleotides. This selective labeling technique enabled us to obtain the chemical shift assignments of these oligonucleotides in complex with the eukaryotic translation initiation factor 4E (eIF4E). Furthermore, we were able to observed intermolecular NOE interactions between the RNA and protein.

show abstract

Section: Discussionmentioning

confidence: 99%

Efficient Synthesis of ¹³C,¹⁵N-Labeled RNA Containing the Cap Structure m⁷GpppA

et al. 2000

Self Cite

View full text Add to dashboard Cite

show abstract

“…Accordingly, 4,4‘,4‘ ‘-tris(benzoyloxy)trityl (TBTr) , was selected to meet these criteria. The TBTr group has been previously used for protection of the primary hydroxyl group of deoxynucleosides 38 and for exo -amino protecting group of deoxyadenosine, deoxycytidine and deoxyguanosine in DNA 39 and RNA synthesis.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Properties of Aminoacylamido-AMP: Chemical Optimization for the Construction of an N-Acyl Phosphoramidate Linkage

et al. 2000

Self Cite

View full text Add to dashboard Cite

This paper describes the design and synthesis of a new type of aminoacyl-adenylate analogue (aa-AMPN) having an N-acyl phosphoramidate linkage where the oxygen atom of the mixed anhydride bond of aminoacyl-adenylate (aa-AMP) is replaced by an amino group. This new type of aa-AMP analogue is expected to be useful as material for studies on the recognition mechanism of the aminoacylation of tRNA and other biochemical reactions. The condensation of phosphoramidite derivatives of carboxamides with nucleoside derivatives failed, because the activated phosphoramidite derivatives reacted with not only the hydroxyl groups but also another reactive species. An alternative approach was examined by the reaction of 5'-O-phosphoramidite adenosine derivatives with carboxamide derivatives. The TBTr and TSE groups were chosen for protection of the amino group of amino acid amides and the phosphate group, respectively. Detailed studies revealed that the use of 5-(3,5-dinitrophenyl)-1H-tetrazole as an activating catalyst of phosphoramidites resulted in rapid condensation within 10 min to give fully protected aa-AMPN derivatives. No side reaction occurred. Deprotection of these products via a two-step procedure gave aa-AMPN derivatives in good yields. It also turned out that aa-AMPNs thus obtained are stable under both acidic and basic conditions, such as 0.1 M HCl (pH 1.0) and 0.1 M NaOH (pH 13.0).

show abstract

“…Two other examples of synthetic capped RNA of much longer sequence (42-mer and 130-mer) have been reported. The 42-mer was synthesized in three fragments that were enzymatically ligated (Iwase et al 1992). The 130mer sequence was chemically assembled by using 2-cyanoethoxymethyl as the 29-OH protecting group, then the 59 end was pyrophosphorylated to add the cap structure with a capping enzyme (Nagata et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase

Thillier¹,

Decroly²,

Morvan³

et al. 2012

RNA

View full text Add to dashboard Cite

The 59 end of eukaryotic mRNA carries a N 7 -methylguanosine residue linked by a 59-59 triphosphate bond. This cap moiety ( 7m GpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 59 exonucleases and avoiding its recognition as ''nonself'' by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 59-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N 7 -methylguanosine, the synthesis of RNAs possessing a cap structure at the 59 end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 29-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 59-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN 29-Om -RNAs were purified before the N 7 -methyl group was added by enzymatic means using the human (guanine-N 7 )-methyl transferase to yield 7m GpppN-RNAs (cap-0) or 7m GpppN 29-Om -RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies.

show abstract

Molecular design of a eukaryotic messenger RNA and its chemical synthesis

Cited by 22 publications

References 47 publications

Efficient Synthesis of ¹³C,¹⁵N-Labeled RNA Containing the Cap Structure m⁷GpppA

Efficient Synthesis of ¹³C,¹⁵N-Labeled RNA Containing the Cap Structure m⁷GpppA

Synthesis and Properties of Aminoacylamido-AMP: Chemical Optimization for the Construction of an N-Acyl Phosphoramidate Linkage

Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase

Contact Info

Product

Resources

About

Molecular design of a eukaryotic messenger RNA and its chemical synthesis

Cited by 22 publications

References 47 publications

Efficient Synthesis of 13C,15N-Labeled RNA Containing the Cap Structure m7GpppA

Efficient Synthesis of 13C,15N-Labeled RNA Containing the Cap Structure m7GpppA

Synthesis and Properties of Aminoacylamido-AMP: Chemical Optimization for the Construction of an N-Acyl Phosphoramidate Linkage

Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase

Contact Info

Product

Resources

About

Efficient Synthesis of ¹³C,¹⁵N-Labeled RNA Containing the Cap Structure m⁷GpppA

Efficient Synthesis of ¹³C,¹⁵N-Labeled RNA Containing the Cap Structure m⁷GpppA

Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase