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mRNA display, like phage display, plavix prescription assistance is a technique used to develop proteins that have a high affinity for specific targets. mRNA display is widely employed in the field of pharmacology to discover new drugs, identify new drug delivery mechanisms, and improve efficacy.
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The ribosome is the link between the genotype and phenotype of an organism. It is the midway point between the genetic code, RNA and the functioning protein. The mRNA technique is often chosen over phage display as it allows the production of longer and more sophisticated peptides. Phage display is also limited by the length of proteins that can be achieved, with a maximum length of approximately one billion amino acids.
In mRNA display, proteins can reach several greater orders of magnitude, up to a theoretical 1015 amino acids in size. This enables the discovery of potentially effective proteins.
Principles of ribosome display
Ribosome display is a self-read technology for in vitro selection and evolution of proteins encoded by DNA libraries. During the process, the protein phenotypes are linked physically to their corresponding mRNA genotypes in stable protein-ribosome mRNA (PRM) complexes. It is used to select high affinity specific antibody ligand binding peptides and other antibody proteins.
Methodology: DNA construct preparation
The process begins with a DNA library. This is a collection of a series of DNA segments that correspond to a variety of potentially useful proteins in solution. Next, the selected DNA fragment is amplified by polymerase chain reaction (PCR).
The resultant DNA fragments are ligated into an expression vector referred to as display construct, or DNA cassette which initiates translation at the target gene promoter. A strong promoter, such as phage promoter T7 ensures robust transcription. This is followed by a spacer sequence which stabilizes the protein construct.
Ternary construct production and affinity chromatography
The prepared construct is subsequently transcribed in vitro. Crucially, the mRNA does not contain a stop codon which results in tethering of the mRNA to the translated protein, with the ribosome serving as a connector.
The spacer sequence stabilizes the attachment of the protein and mRNA to the ribosome. The ternary mRNA–ribosome–protein complexes then undergo affinity chromatography. This separation technique allows potential binding targets to be immobilised on a solid medium, while a solution containing the ternary complex is applied. The protein corresponding to the library member encoding it will bind if it displays high affinity to the target. Washing of the column eliminates poor or non-specific binding of the complex to targets.
The high affinity ternary complexes are subsequently eluted, and the mRNA of the bound complexes is obtained following their dissociation from the connecting ribosomes. The eluted complexes deliver a protein that binds with high affinity for its target.
mRNA isolation and reverse transcription
The isolated mRNAs are then subject to reverse transcription. In this process, the mRNA generates the initial DNA molecule that encodes it–hence the term reverse. This is accomplished via reverse transcriptase enzyme and the DNA produced is called cDNA (copy DNA) which contains the full sequence corresponding to the protein. The reason for this step is because mRNA has a short half-life; it is highly unstable in solution compared to DNA.
The cDNA is subject to mutagenesis, which slightly alters the resultant protein structure. As such, the cDNA is continually subject to a selective process which allows simulation of the natural selection in vitro to achieve high affinity protein binding.
Theoretically, the library size in phage display can accommodate 1×1014, thus the population of potential peptide and protein candidates is significantly large. This increases the probability of candidate discovery across drugs, antibodies and other therapeutic proteins.
- All RNA Content
- What is RNA?
- RNA Structure
- Types of RNA: mRNA, rRNA and tRNA
- RNA Synthesis
Last Updated: Feb 26, 2019
Hidaya is a science communications enthusiast who has recently graduated and is embarking on a career in the science and medical copywriting. She has a B.Sc. in Biochemistry from The University of Manchester. She is passionate about writing and is particularly interested in microbiology, immunology, and biochemistry.
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