Previously, the in vitro production of mRNA was limited in yield and translational activity by the co-transcriptional capping method using cap analog.The presence of dinucleotide cap analog in the transcription reaction mix greatly decreases the mRNA yields and inhibits translation when non-incorporated dinucleotide analogs are not carefully removed. Instead of using a costly and inefficiently incorporated nucleotide cap analog, the C3P3® mRNA Production System uses a virus-derived capping enzyme physically coupled to the RNA polymerase to add the cap at high efficiency capping - versus 80% for other systems using standard m7GpppG cap analog or anti-reverse cap analog (ARCA) and 100% proper cap orientation. The standard cap analog (m7GpppG) used in co-transcriptional reactions can be incorporated in the reverse orientation (33-50% of the time) which is not translated in the cell, thereby decreasing the amount of protein produced per microgram of transfected mRNA.. Simultaneously, the capped ...
This methylated ribonucleotide can be incorporated onto the 5´-end of in vitro transcripts and simulates the cap structure found on most eukaryotic mRNA molecules.
Due to the complexity of preparing modRNA and the high cost of its reagents, there is a need for an improved, cost-efficient protocol to make modRNA. Here we show that changing the ratio between anti-reverse cap analog (ARCA) and N1-methyl-pseudouridine (N1mΨ), favoring ARCA over N1mΨ, significantly increases the yield per reaction, improves modRNA translation, and reduces its immunogenicity in vitro. This protocol will make modRNA preparation more accessible and financially affordable for basic and translational research.. ...
New RNA cap analogs are disclosed containing one or more phosphorothioates groups. The analogs also contain modifications at the 2′-O position of 7-methylguanosine that prevent them from being incorporated in the reverse orientation during in vitro synthesis of mRNA and that hence are
Interacting selectively and non-covalently with a hypermethylated cap structure consisting of 7-methylguanosine (m(7)G) followed by four methylated nucleotides (cap 4): 7-methylguanosine-ppp-N6, N6, 2-O-trimethyladenosine-p-2-O-methyladenosine-p-2-O-me…
Blocking of the 3´ -hydroxyl of m7G with 3´-0-Me assures that the capped transcripts are homogeneous. The 3´ - hydroxyl of the non-methylated G is the only 3´ - hydroxyl available for initiation.
Friedrich A , Wildner M , Tillmanns E , Merz P L , American Mineralogist , 85 (2000) p.593-599, Crystal chemistry of the new mineral brandholzite, Mg(H2O)6[Sb(OH)6]2,, and of the synthetic analogues M(H2O)6[Sb(OH)6]2 (Me=Mg,Co) ...
I agree that we have covered the bases in terms of abilities. We have 2 useful and complementary abilities already; any more, and this CAP will become too...
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The eukaryotic cap-binding complex eIF4F is an essential component of the translational machinery. Recognition of the mRNA cap structure through its subunit eIF4E is a requirement for the recruitment of other translation initiation factors to the mRNA 5-end and thereby for the attachment of the 40 S ribosomal subunit. In this study, we have investigated the mechanistic basis of the observation that eIF4E binding to the cap is enhanced in the presence of the large eIF4F subunit, eIF4G. We show that eIF4E requires access to both the mRNA 5-cap and eIF4G to form stable complexes with short RNAs. This stabilization can be achieved using fragments of eIF4G that contain the eIF4E binding site but not the RNA recognition motifs. Full-length eIF4G is shown to induce increased eIF4E binding to cap analogues that do not contain an RNA body. Both results show that interaction of eIF4G with the mRNA is not necessary to enhance cap binding by eIF4E. Moreover, we show that the effect of binding of ...
The 5´terminal m7G cap present on most eukaryotic mRNAs promotes translation in vitro at the initiation level. For most RNAs, elimination of the cap structure causes a loss of stability, especially against exonuclease degradation, and a decrease in the formation of the initiation complex of mRNAs for protein synthesis. Certain prokaryotic mRNAs containing a 5´ terminal cap structure are translated as efficiently as or more efficiently than eukaryotic mRNAs in a eukaryotic cell-free protein synthesizing system. Also a cap requirement has been observed for splicing eukaryotic substrate RNAs. A method for efficient in vitro synthesis of capped RNA using E. coli RNA polymerase primed with m7G(5´ )ppp(5´ )G or m7G(5´ )ppp(5´ )A has been developed by Contreas et al.
15-3 Cap Structure Purified cap by DEAE chromatography; digested with enzymes to figure structure  -phosphate of NTP remains only in first nucleotide in RNA -Cap is 5-terminus of RNA -Cap is m 7 G, 7- methylguanosine, -Linkage is triphosphate -Charge on cap area is ~ -5 Fig. 1 vaccinia virus 3H methyl from S-AdoMet; 32P-GTP label RNA); KOH to digest RNA Fig. 2 shows me7-G
EC 3.6.1.59 Accepted name: 5-(N7-methyl 5-triphosphoguanosine)-[mRNA] diphosphatase. Reaction: a 5-(N7-methyl 5-triphosphoguanosine)-[mRNA] + H2O = N7-methylguanosine 5-phosphate + a 5-diphospho-[mRNA]. Other name(s): DcpS; m7GpppX pyrophosphatase; m7GpppN m7GMP phosphohydrolase; m7GpppX diphosphatase; m7G5ppp5N m7GMP phosphohydrolase. Systematic name: 5-(N7-methyl 5-triphosphoguanosine)-[mRNA] N7-methylguanosine 5-phosphate phosphohydrolase. Comments: The enzyme removes (decaps) the N7-methylguanosine 5-phosphate cap from an mRNA degraded to a maximal length of 10 nucleotides [3,6]. Decapping is an important process in the control of eukaryotic mRNA degradation. The enzyme functions to clear the cell of cap structure following decay of the RNA body [2]. The nematode enzyme can also decap triply methylated substrates, 5-(N2,N2,N7-trimethyl 5-triphosphoguanosine)-[mRNA] [4].. Links to other databases: BRENDA, EXPASY, KEGG, Metacyc, CAS registry number: References:. 1. Malys, N. and ...
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What is the difference between Prokaryotic and Eukaryotic mRNA? Prokaryotic mRNA is polycistronic while eukaryotic mRNA is monocistronic. In prokaryotic mRNA,..
The 5′ terminal cap structure, m7GpppX (where X is any nucleotide), is a ubiquitous feature of eukaryotic mRNAs that plays an important role in the cytoplasm and the nucleus. In the cytoplasm the cap...
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displaystyle \paren {E_0 \cap E_1 \cap E_2 \cap \ldots} \cup \paren {E_1 \cap E_2 \cap E_3 \cap \ldots} \cup \cdots ...
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To ensure that only RNAP II transcripts are capped, the RNAP II CTD is phosphorylated, and the phosphorylated CTD recruits capping enzymes. After ~25-30 nucleotides have been transcribed and the nascent pre-mRNA is emerging from RNAP, the first transcribed nucleotide (the 5 end) is bound via a 5-5 triphosphate linkage to a 7-methylguanosine. The 2-hydroxyls of the first two transcribed nucleotides are also often methylated. ...
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Looking for cap-binding protein? Find out information about cap-binding protein. A protein that specifically recognizes the methylated cap of eukaryotic messenger ribonucleic acid and is essential in the regulation of mRNA translation Explanation of cap-binding protein
Degradation of mRNA is a highly regulated step important for proper gene expression. Degradation of eukaryotic mRNA is initiated by shortening of the 3 end located poly(A) tail. Poly(A)-specific ribonuclease (PARN) is an oligomeric enzyme that degrades the poly(A) tail with high processivity. A unique property of PARN is its ability to interact not only with the poly(A) tail but also with the 5 end located mRNA cap structure. A regulatory role in protein synthesis has been proposed for PARN based on its ability to bind the cap that is required for efficient initiation of eukaryotic mRNA translation. Here we have investigated how the cap structure influences PARN activity and how PARN binds the cap. We show that the cap activates PARN and enhances the processivity of PARN. Further we show that the cap binding complex (CBC) inhibits PARN activity through a protein-protein interaction. To investigate the cap binding property of PARN, we identified the cap binding site at the molecular level using ...
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Using these BPC bags with 3 ports (MPC insert, MPC body, luer lock), simplify cleaning and disposal and reduce cross-contamination risks for 2 to 50L volumes of media, buffers, water for injection (WFI) and biological products. Labtainer: End Ported with QC Insert and Body andFemale LL , 20 L ...