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DNA Library Preparation

mRNA display serves as an in vitro methodology utilized for exploring natural or artificially created DNA libraries to identify the operational proteins and peptides they carry. CD BioSciences offers an mRNA display-based platform with libraries containing trillions of diverse peptides to identify effective peptide binders tailored to your specific target. Our services encompass the design and construction of libraries to aid in the successful execution of subsequent experiments.

Peptide Selection Using mRNA Display- CD BioSciences Fig 1. General scheme for peptide selection by mRNA display.

Strategies for DNA Library Preparation

The process of constructing an mRNA display library starts with the creation of a DNA library. The primary consideration when commencing the selection of proteins through mRNA display involves formulating and assembling the library at the DNA level. A DNA collection for any desired protein or minor peptide can be generated through solid-phase synthesis, succeeded by PCR amplification.

The T7 promoter and TMV translation enhancer sequences are necessary for transcription initiation and efficient translation.
The DNA library will ultimately need to encode terminal constant regions to permit PCR amplification.
These may also encode protein affinity tags that will facilitate the purification of the resulting displayed proteins.
Including nearby restriction sites aids in modifying constant regions near random sequences for potential library reengineering.
The puromycin link site is complementary to the puromycin-containing oligonucleotide.

DNA library design. Fig 2. DNA library design for an mRNA display selection. (Seelig, B., 2011)

Methods of DNA Library Construction

Methods	Description
Synthetic Method	Utilize chemical synthesis methods to create a DNA library with multiple variant sites, allowing precise control over the type and frequency of each mutation.
Enzymatic Cleavage and Ligation	Cut DNA fragments in the DNA library using restriction endonucleases and then reassemble these fragments with ligases to create a DNA library with diverse sequences.
Genomic Library Approach	Extract DNA fragments from the genome to construct a DNA library, ensuring that the library contains a significant portion of genomic information.

Our Services

CD BioSciences uses a synthetic method to construct the DNA library for mRNA display. We utilize NNG/C codons to encode random regions. The length of peptides in DNA libraries can be regulated by controlling the number of NNS.

Fig 3. Synthetic DNA library.

Generating Macrocyclic Peptide Library

mRNA display enables the effective identification of cyclic peptides. The library's structure is crucial for subsequent cyclic peptide screening via mRNA display. Cyclization is achieved through chemical synthesis. We employ a method that cyclizes two cysteine residues via meta-substituted dibromo-xylene, forming a cyclic peptide library with irreducible disulfide bonds.

Fig 4. Generation of macrocyclic peptides by mRNA display.

Library Construction Services

Our DNA library preparation services for mRNA display include the generation of synthetic peptide and natural proteome libraries as well as the steps required for generation of mRNA–protein fusion libraries. Our goal is to provide you with peptide development services quickly and with high quality.

CD BioSciences' mRNA display-based peptide discovery services is meticulously designed to cater to your research needs with precision and excellence. Partner with us for unparalleled expertise and support in early drug discovery. contact us for more information.

References

Seelig, B. mRNA display for the selection and evolution of enzymes from in vitro-translated protein libraries. Nature Protocols. 2011, 6(4): 540-552.
Peacock, H., Suga, H. Discovery of de novo macrocyclic peptides by messenger RNA display. Trends in Pharmacological Sciences. 2021, 42(5): 385-397.

For Research Use Only. Not For Clinical Use.