Enzymatic RNA capping method
An RNA capping, methyltransferase technology, applied in chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve problems such as inefficiency
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Embodiment 1
[0078] Example 1: RNA capping activity of H3C2 and VCE at high temperature
[0079] In 10 μL of 1× RNA capping buffer (50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM DTT, pH 8.0), in the presence of 0.5 mM GTP and 0.1 mM SAM in the presence of 0.1 mM SAM, 10 nM purified VCE or H3C2 RNA capping enzyme was mixed with 500 nM RNA1 ( figure 1 ) for 30 minutes, RNA1 contains a fluorescent group (FAM) at its 3' end. The reaction was quenched by adding 1 μL of 50 mM EDTA followed by heat inactivation at 70° C. for 10 minutes. Quenched reactions were analyzed by capillary electrophoresis using an Applied Biosystem 3730xl Genetic Analyzer. The peak areas corresponding to m7Gppp-, unmethylated Gppp-, uncapped pp- and ppp-RNA1 were quantified and used to calculate the fraction of m7Gppp- at the end of the 30 min capping reaction. exist figure 2 In , each bar represents the mean of 4 independent experiments. Error bars represent standard deviation of 4 replicates. It is obvious that...
Embodiment 2
[0085] Example 2: Identification and testing of H3C2 orthologues
[0086] Under in vitro conditions, from faustovirus D5b ( Purified RNA capping enzyme H3C2 with accession number AMN83561) is active up to 60°C. Through sequence homology, several putative orthologs in other faustovirus strains were identified. Gene products of putative RNA capping enzymes from other faustovirus strains and the related giant viruses Acanthamoeba mimivirus and Acanthamoeba mumuvirus were found to be effective in RNA capping up to 55-60°C active. The ORF of the identified orthologous gene was synthesized and inserted into a T7 expression vector, so that the expression of the target gene was under the control of the T7 promoter. According to the manufacturer's recommendations, use High-fidelity 2× Master Mix (New England Biolabs, Ipswich, MA) amplified the T7 expression cassette by PCR. According to the manufacturer's instructions, use The PCR & DNA cleanup kit (New England Biolabs, Ipsw...
Embodiment 3
[0087] Example 3: Sequence Analysis
[0088] Sequence alignment analysis showed that among the four Faustovirus RNA capping enzymes active up to 55°C, three regions of 60 amino acids or 111 amino acids in length showed 90% or higher amino acid sequence identity ( Figure 5 ). The conserved region Fausto_CR_01 spans 60 amino acids within the TPase domain of H3C2. Fausto_CR_03 spans 111 amino acids at the N-terminus of the guanosine N7 methyltransferase domain. Fausto_CR_05 spans 60 amino acids on the C-terminal side of the guanosine N7 methyltransferase domain of H3C2. The amino acid sequence of Fausto_CR_01 (which corresponds to amino acids 43-102 of the H3C2 sequence) is FDKLKPDGEITTTMRVSNADGMAREITFGGGVKTGEMFVKKQNICVFDVVDIFSYKVAVS (SEQ ID NO: 2). The amino acid sequence of Fausto_CR_03 (which corresponds to amino acids 537-647 of the H3C2 sequence) is GFYGNNYKIASDIYLNYIDVFNFDDLWKYNPGYFEKNKSDIYIAPNKYRRYLIKSLFNKYIKNAKWVIDAAAGRGADLHLYKAECVENLLAIDIDPTAISELIRRRNEITG (SEQ ID N...
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