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The technique of mRNA display has gained popularity for screening large libraries of peptides or proteins. Utilizing the advantages of mRNA display, it has emerged as a key strategy for generating peptides and proteins with antibody affinity. At CD BioSciences, we leverage the innovative power of mRNA display technology to accelerate and streamline the drug development process.
Fig 1. Structure of tyrosyl tRNA and Puromycin. (Huang, et al., 2018)
mRNA display is a selective technique where protein variants form covalent bonds with their encoding mRNA molecules. The covalent bond between mRNA and the corresponding nascent peptide relies on the puromycin connection, utilizing the mRNA-DNA-puromycin fusion as a template for in vitro translation. Puromycin, a structural mimic of tyrosyl tRNA, disrupts translation, leading to premature product release.
Fig 2. Techniques for the attachment of the puromycin linker for mRNA display.(Huang, et al., 2018)
Enhancing the efficiency of mRNA linker attachment is crucial for improving the generation efficiency of mRNA-puromycin fusion. The length and sequence of the linker play a significant role in influencing the fusion peptide's production. Prior to mRNA display gaining wider adoption, enhancing the synthesis of the mRNA-puromycin conjugate was imperative.
Fig 3. General scheme for peptide selection by mRNA display.
To overcome this issue, different methods such as splint ligation, photo-cross-linking ligation, and the Y-ligation method have been developed. The main difference in these methods lies in the structure of the puromycin linker that bridges mRNA and protein.
| Methods | Description |
| UV crosslinking | The puromycin linker can be added to the mRNA using photo-crosslinking by incorporating a psoralen. Excitation of psoralen by light irradiation causes it to react with the puromycin molecule and form a stable linker. |
| Splint ligation | Ligation by splint ligation using a strand of DNA complementary to both mRNA and puromycin linker sequence. |
| Y-ligation | The method involves the use of T4 RNA ligase, which can join complementary RNA and DNA strands using ATP. |
| TRAP system | Added puromycin linker anneals to the complementary portion of the mRNA. Upon reaching a stop codon during translation puromycin enters the ribosomal A site and is covalently attached to the translated peptide. |
CD BioSciences utilizes the Y-ligation technique for efficient joining, linking the single-stranded mRNA 3' end with the phosphorylated 5' end of the puromycin-FITC DNA tag. Our custom-designed DNA tag, incorporating puromycin, forms a ligation with the transcribed mRNA, resulting in the ligated mRNA-tag product. This product is employed in in vitro translation processes.
Fig 4. Schematic of the puromycin-tag ligation process known as Y-ligation.
Template Preparation
Reaction was set up consisting of final concentrations of 4 μM transcribed mRNA, 6 µM puromycin-FITC DNA tag, and 1 mM ATP in T4 ligation buffer.
Annealing
The reaction was incubated at 90°C for 30 s and cooled to room temperature with a 1°C/s decline rate to correctly anneal the mRNA library with the puromycin-FITC DNA tag.
Ligation
T4 PNK Kinase and T4 RNA ligase were added to the reaction mixture and further incubated at 25°C for 30 min.
Gel Electrophoresis
Transcribed mRNA libraries and their DNA tag ligated products were resolved using mini size 8 M Urea 6% polyacrylamide Tris-borate-EDTA gel at constant 50 mA.
Purification and Recovery
To eliminate unligated products, ligated mRNA-tag product was further purified through gel electrophoresis and recovered using electroelution followed by ethanol precipitation.
At CD BioSciences, we are dedicated to transforming drug development through innovative mRNA display technology. To learn more about how we can support your drug development efforts with our mRNA display services, please contact us. Our team is ready to discuss your project requirements and design a customized solution that meets your needs.
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