Anti reverse cap analog

Manufactured by TriLink

Sourced in United States

The Anti-Reverse Cap Analog is a laboratory equipment product designed to prevent the reverse rotation of caps or closures during the opening and closing of various containers. It serves as a mechanical solution to secure caps and maintain their proper orientation.

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Anti-Reverse Cap Analog 3′-O-Me-m7G(5′)ppp(5′)G (2 citations) Anti-Reverse Cap Analog (ARCA) capped and polyadenylated AsCpf1 mRNA and LbCpf1 mRNA transcripts (2 citations)

14 protocols using anti reverse cap analog

Radium-1 TCR mRNA Synthesis

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A TGFβRII frameshift mutation-specific, human leukocyte antigen (HLA)-A2 restricted TCR was identified in a T cell clone from a vaccinated MSI+ colon cancer patient and named Radium-1 [18 (link)]. The in vitro mRNA synthesis was performed essentially as previously described [20 (link)]. Anti-Reverse Cap Analog (Trilink Biotechnologies Inc., USA) was used for RNA capping. The mRNA quality was assessed by agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific, USA).

Mensali N., Myhre M.R., Dillard P., Pollmann S., Gaudernack G., Kvalheim G., Wälchli S, & Inderberg E.M. (2019). Preclinical assessment of transiently TCR redirected T cells for solid tumour immunotherapy. Cancer Immunology, Immunotherapy, 68(8), 1235-1243.

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Synthesis and Purification of mRNA

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All mRNAs were synthesized using MegaScript T7 Transcription Kit (Thermo Fisher Scientific, MA, USA) with kit-supplied rATP, rCTP, rUTP, 1:4 premix of rGTP, and Anti Reverse Cap Analog (TriLink Biotechnologies, CA, USA). The reaction mixtures were incubated at 37°C for 4 hours and further incubated at 37°C for 30 minutes in the presence of TURBO DNase (Thermo Fisher Scientific, MA, USA). RNA products were purified with an RNA extraction column (Favorgen Biotect, Taiwan) according to the manufacturer’s protocol and then subjected to treatment with Antarctic Phosphatase (NEB, MA, USA) at 37°C for 30 minutes. Finally, the product mRNAs were purified again using RNeasy MiniElute Cleanup Kit (Qiagen, Germany). The amounts of product mRNAs were determined by NanoVue (GE Healthcare, UK). The purity of mRNAs was assessed using UREA-PAGE following the standard protocol using 5% gel, stained with GelRed Nucleic Acid Gel Stain (Biotium, CA, USA), and the images were taken by GelDoc Go Imaging System (Bio-Rad, CA, USA). mRNAs were diluted to 100 ng/μL in water and stored at −20°C until use.

Li C.Y., Liang Z., Hu Y., Zhang H., Setiasabda K.D., Li J., Ma S., Xia X, & Kuang Y. (2022). Cytidine-containing tails robustly enhance and prolong protein production of synthetic mRNA in cell and in vivo. Molecular Therapy. Nucleic Acids, 30, 300-310.

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In Vitro Transcription of Modified mRNAs

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All mRNAs were generated using the above PCR products and MEGAscript T7 Kit (Ambion, USA). In the reaction, pseudouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate (TriLink BioTechnologies, USA) were used instead of uridine triphosphate and cytosine triphosphate, respectively. For IVT of the MS2CP-responsive mRNA used in Fig. 3, N1-methylpseudouridine-5′-triphosphate (m1pU) (TriLink BioTechnologies) was used instead of uridine-5′-triphosphate. Guanosine-5′-triphosphate was 5-fold diluted with an anti-reverse cap analog (TriLink BioTechnologies) before the IVT reaction. Reaction mixtures were incubated at 37°C for up to 6 h and then mixed with TURBO DNase (Ambion), and further incubated at 37°C for 30 min to remove the template DNA. The resulting mRNAs were purified using a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech, Taiwan), incubated with Antarctic Phosphatase (New England Biolabs) at 37 °C for 30 min, and then purified again using an RNeasy MinElute Cleanup Kit (QIAGEN).

Matsuura S., Ono H., Kawasaki S., Kuang Y., Fujita Y, & Saito H. (2018). Synthetic RNA-based logic computation in mammalian cells. Nature Communications, 9, 4847.

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Synthesis of Modified mRNA Transcripts

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All mRNAs were synthesized using MegaScript T7 Kit (ThermoFisher Scientific). Native mRNAs were synthesized with kit-supplied ATP, CTP, UTP, 1:4 premix of GTP and Anti Reverse Cap Analog (TriLink BioTechnologies). Ψ (pseudouridine-5′-triphosphate), m1Ψ (N¹-methylpseudouridine-5′-triphosphate), m6A (N⁶-methyladenosine-5′-triphosphate) and m5C (5-methylcytidine-5′-triphosphate) were purchased from TriLink Biotechnologies and used as full substitutions of UTP, ATP or CTP in the reaction to synthesize modified mRNAs. The reaction mixtures were incubated at 37 °C for 4 h and further incubated at 37 °C for 30 min in the presence of TURBO DNase (ThermoFisher Scientific). RNA products were purified with a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech) according to the manufacturer's protocol and then treatment with Antarctic Phosphatase (New England Biolabs) at 37 °C for 30 min. For m6A-containing mRNAs, the polyA tails were added using polyA polymerase (New England Biolabs). The final RNA products were purified using RNeasy MiniElute Cleanup Kit (Qiagen) and stored as 100 ng/μl solution in water at −30 °C.

Parr C.J., Wada S., Kotake K., Kameda S., Matsuura S., Sakashita S., Park S., Sugiyama H., Kuang Y, & Saito H. (2020). N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells. Nucleic Acids Research, 48(6), e35.

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Synthesizing Modified mRNA and sgRNA for CRISPR-Cas9

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Cas9 mRNAs (with or without miRNA target sequences and with kink-turn motif), L7Ae mRNAs (with or without miRNA target sequences) and BFP mRNA (without miRNA target sequences) were prepared by using a MEGAscript kit (Ambion). In order to reduce the interferon response caused by long RNA, pseudouridine-5΄-triphosphate and 5-methylcytidine-5΄-triphosphate (TriLink Bio Technologies) were used instead of natural rUTP and rCTP, respectively (18 (link)). Guanosine-5΄-triphosphate was 5-fold diluted with an Anti Reverse Cap Analog (TriLink Bio Technologies) before the IVT reaction. The sgRNA was constructed using a MEGAshortscript kit (Ambion) according to the instruction manual. Because sgRNA with modified bases may cause downregulation of Cas9 activity, natural rNTPs were used for preparing sgRNA. The template DNA was degraded by TURBO DNase (Ambion), and the mRNAs and sgRNA were purified using a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech) incubated with Antarctic Phosphatase (New England Biolabs) at 37°C for 30 min and then purified again using an RNeasy MinElute Cleanup Kit (QIAGEN). For further purification, sgRNA was electrophoresed, extracted from gel (10% polyacrylamide gel, 8.3 M urea), and ethanol-precipitated.

Hirosawa M., Fujita Y., Parr C.J., Hayashi K., Kashida S., Hotta A., Woltjen K, & Saito H. (2017). Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch. Nucleic Acids Research, 45(13), e118.

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Modified mRNA Synthesis and Purification

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mRNAs were generated from the corresponding DNA templates using a MEGAscript T7 Kit (Ambion) and a modified protocol. Psuedouridine-5’-triphosphate and 5-methylcytidine-5’-triphosphate (TriLink BioTechnology) were used instead of uridine-5’-triphosphate and cytidine-5’-triphosphate. A mixture of Anti-Reverse Cap Analog (TriLink) and guanosine-5’-triphosphate (4:1) was used instead of guanosine-5’-triphosphate. Reaction mixtures were incubated at 37 °C for 4 h and then treated with TURBO DNase (Ambion) at 37 °C for 30 min. The RNAs were purified using a FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech) and then treated with Antarctic Phosphatase (New England Biolabs) at 37 °C for 30 min. The resulting mRNAs were purified using an RNeasy MinElute Cleanup Kit (QIAGEN).

Shibata T., Fujita Y., Ohno H., Suzuki Y., Hayashi K., Komatsu K.R., Kawasaki S., Hidaka K., Yonehara S., Sugiyama H., Endo M, & Saito H. (2017). Protein-driven RNA nanostructured devices that function in vitro and control mammalian cell fate. Nature Communications, 8, 540.

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Synthesis of Capped Reporter RNA

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The 5′UTRs of reporter RNA were synthesized by GeneArt (Thermo Fisher Scientific). Templates were prepared by Fusion PCR using the 5′UTR variants, ORFs and common 3′UTR with a poly-A tail. Capped mRNAs were prepared by using the MegaScript Kit (Thermo Fisher Scientific) in the presence of an anti-reverse cap analog (TriLink BioTechnologies). Ten microliters of the reaction solution was prepared and incubated at 37 °C. After 6 h of incubation, 1 μL of TURBO DNase (Thermo Fisher Scientific) was added to the reaction solution, mixed by pipetting, and incubated at 37 °C for 30 min. RNA products were purified with Zymo RNA Clean and Concentrator (Zymo Research). Then, to remove the phosphate at the 5′ terminus, the RNA products were treated with Antarctic Phosphatase (New England Biolabs). The RNA products were purified again, and the concentrations were measured with a NanoDrop 2000 (Thermo Fisher Scientific). Sequence information regarding all DNA materials for constructing the templates of reporter mRNA is available in Supplementary Data 1.

Komatsu K.R., Taya T., Matsumoto S., Miyashita E., Kashida S, & Saito H. (2020). RNA structure-wide discovery of functional interactions with multiplexed RNA motif library. Nature Communications, 11, 6275.

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In Vitro mRNA Synthesis and Characterization

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The in vitro mRNA synthesis was performed essentially as previously described^{40 (link)}. Anti-Reverse Cap Analog (Trilink Biotechnologies Inc., San Diego, CA, USA) were used to cap the RNA. The mRNA quality was assessed by agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific).

Walseng E., Köksal H., Sektioglu I.M., Fåne A., Skorstad G., Kvalheim G., Gaudernack G., Inderberg E.M, & Wälchli S. (2017). A TCR-based Chimeric Antigen Receptor. Scientific Reports, 7, 10713.

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Synthesis and Purification of PRDX1 and CAR-CD19 mRNA

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mRNA for PRDX1 and CAR-CD19 was synthesized in vitro with the use of the RiboMAX Large-Scale RNA Production System (Promega). The PRDX1 coding sequence was subcloned from PRDX1-pETMM11 plasmid to mRNA expression vector pCIpA102 (kindly provided by Dr. Stein Sæbøe-Larssen from Oslo University Hospital) with the use of FastDigest EcoRI and NotI restriction enzymes (Thermo Fisher Scientific). pCIpA102 plasmid containing CAR-CD19 coding sequence was kindly provided by Dr. Jon Amund Kyte Oslo University Hospital under MTA. Both plasmids pCIpA102-PRDX1 and pCIpA102-CAR-CD19 were linearized with MfeI restriction enzyme (New England BioLabs) and used for the mRNA synthesis reaction. Anti-Reverse Cap Analog (Trilink Biotechnologies) was used to cap the mRNA. The synthesis reaction was carried out for 4 hours at 37°C. Then, RNA-free DNase was added to digest the residual DNA matrix. The synthesized mRNA was then purified using either the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific) or the lithium chloride (LiCl; Thermo Fisher Scientific) precipitation method. The mRNA's quality and quantity were assessed by agarose gel electrophoresis and NanoDrop (Thermo Scientific).

Klopotowska M., Bajor M., Graczyk-Jarzynka A., Kraft A., Pilch Z., Zhylko A., Firczuk M., Baranowska I., Lazniewski M., Plewczynski D., Goral A., Soroczynska K., Domagala J., Marhelava K., Slusarczyk A., Retecki K., Ramji K., Krawczyk M., Temples M.N., Sharma B., Lachota M., Netskar H., Malmberg K.J., Zagozdzon R, & Winiarska M. (2021). PRDX-1 Supports the Survival and Antitumor Activity of Primary and CAR-Modified NK Cells under Oxidative Stress. Cancer Immunology Research, 10(2), 228-244.

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Identifying hTERT-specific TCR in Pancreatic Cancer

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A hTERT-specific, HLA-DP04-restricted TCR was identified in a CD4⁺ T cell clone from a vaccinated pancreatic cancer patient and named Radium-4.²³ (link) The in vitro mRNA synthesis was performed, essentially as previously described.⁶³ (link) Anti-Reverse Cap Analog (Trilink Biotechnologies, San Diego, CA, USA) was used to cap the RNA. The mRNA was assessed by agarose gel electrophoresis and Nanodrop (Thermo Fisher Scientific).

Dillard P., Köksal H., Maggadottir S.M., Winge-Main A., Pollmann S., Menard M., Myhre M.R., Mælandsmo G.M., Flørenes V.A., Gaudernack G., Kvalheim G., Wälchli S, & Inderberg E.M. (2020). Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy. Molecular Therapy, 29(3), 1199-1213.

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