Cap Analog Method
Cap Analog Method - Related Videos
08:55
In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
0 Views •
Cited by 13 •
2012
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
11:15
Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping
0 Views •
Cited by 4 •
2011
The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.
10:40
Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions
0 Views •
Cited by 3 •
2016
Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.
11:00
Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis
0 Views •
Cited by 8 •
2010
Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.
02:48
Labeling of Replicating HSV-1 Genomes with a Nucleoside Analog
0 Views •
2026
Source: Dembowski, J. A., Deluca, N. A. Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J. Vis. Exp. (2017)This video demonstrates the infection of stationary-phase mammalian cells with HSV-1 (Herpes Simplex Virus-1), followed by replication of the viral genome using host and viral replication machinery. A synthetic nucleoside analog is incorporated into newly synthesized viral DNA to label replicating genomes, which are subsequently isolated for...
FAQs
Anti-reverse cap analogs favor incorporation of the synthetic cap in an orientation suitable for recognition by translation machinery. This directional control helps the RNA’s 5′ end more closely resemble the organization expected of mature eukaryotic messenger RNA, supporting more effective use of the transcript in biological systems.
The orientation of the 5′ cap influences how biological systems recognize the transcript. A correctly oriented cap can support the RNA features needed for translation, whereas an unsuitable orientation may reduce that functional resemblance to mature messenger RNA. Anti-reverse cap analogs therefore improve the likelihood that capped transcripts will support efficient protein production.
Nucleotides provide the building blocks for synthesizing the RNA, while the cap analog supplies the synthetic structure intended for the transcript’s 5′ end. Including both during in vitro transcription connects RNA production with cap incorporation in the same reaction, producing a transcript designed for improved stability and translation in biological systems.
The main difference is the presence of a synthetic 5′ cap on the transcript. Compared with uncapped RNA, capped RNA generally remains more stable and undergoes translation more efficiently in biological systems. The method therefore changes the functional behavior of an in vitro transcript, not merely its chemical composition.
A researcher performs in vitro transcription using the RNA template, nucleotides, and a cap analog supplied in the reaction. When appropriate orientation is important, an anti-reverse cap analog can be selected to promote correct incorporation at the 5′ end. The resulting capped transcript is then used in a biological system for analysis or protein production.
This approach is useful when investigators need in vitro-produced RNA to function more like mature eukaryotic messenger RNA. Supported applications include studying gene expression and mRNA function, producing protein from synthetic transcripts, and developing RNA-based experimental platforms. Its value comes from combining controlled RNA production with improved stability and translation.