MRNA display

Last updated

mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target. The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage. The complex then binds to an immobilized target in a selection step (affinity chromatography). The mRNA-protein fusions that bind well are then reverse transcribed to cDNA and their sequence amplified via a polymerase chain reaction. The result is a nucleotide sequence that encodes a peptide with high affinity for the molecule of interest.

Contents

Puromycin is an analogue of the 3’ end of a tyrosyl-tRNA with a part of its structure mimics a molecule of adenosine, and the other part mimics a molecule of tyrosine. Compared to the cleavable ester bond in a tyrosyl-tRNA, puromycin has a non-hydrolysable amide bond. As a result, puromycin interferes with translation, and causes premature release of translation products.

Figure 1. mRNA-Polypeptide Fusion Formation. a. Ribosome moves along the mRNA template and nascent peptide is being made. When the ribosome reaches the 3' end of the template, the fused puromycin will enter the A site of the ribosome. b. The mRNA-polypeptide fusion is released. Figure 1. mRNA-Polypeptide Fusion Formation.png
Figure 1. mRNA-Polypeptide Fusion Formation. a. Ribosome moves along the mRNA template and nascent peptide is being made. When the ribosome reaches the 3’ end of the template, the fused puromycin will enter the A site of the ribosome. b. The mRNA-polypeptide fusion is released.

All mRNA templates used for mRNA display technology have puromycin at their 3’ end. As translation proceeds, ribosome moves along the mRNA template, and once it reaches the 3’ end of the template, the fused puromycin will enter ribosome’s A site and be incorporated into the nascent peptide. The mRNA-polypeptide fusion is then released from the ribosome (Figure 1).

To synthesize an mRNA-polypeptide fusion, the fused puromycin is not the only modification to the mRNA template. [1] Oligonucleotides and other spacers need to be recruited along with the puromycin to provide flexibility and proper length for the puromycin to enter the A site. Ideally, the linker between the 3’ end of an mRNA and the puromycin has to be flexible and long enough to allow the puromycin to enter the A site upon translation of the last codon. This enables the efficient production of high-quality, full-length mRNA-polypeptide fusion. Rihe Liu et al. optimized the 3’-puromycin oligonucleotide spacer. They reported that dA25 in combination with a Spacer 9 (Glen Research), and dAdCdCP at the 5’ terminus worked the best for the fusion reaction. They found that linkers longer than 40 nucleotides and shorter than 16 nucleotides showed greatly reduced efficiency of fusion formation. Also, when the sequence rUrUP presented adjacent to the puromycin, fusion did not form efficiently. [2]

In addition to providing flexibility and length, the poly dA portion of the linker also allows further purification of the mRNA-polypeptide fusion due to its high affinity for dT cellulose resin. [3] The mRNA-polypeptide fusions can be selected over immobilized selection targets for several rounds with increasing stringency. After each round of selection, those library members that stay bound to the immobilized target are PCR amplified, and non-binders are washed off.

Method

Figure 2. Splint Aid Single Stranded mRNA/DNA T4 DNA Ligase Ligation Figure 2. T4 DNA Ligase Ligation.png
Figure 2. Splint Aid Single Stranded mRNA/DNA T4 DNA Ligase Ligation

The synthesis of an mRNA display library starts from the synthesis of a DNA library. A DNA library for any protein or small peptide of interest can be synthesized by solid-phase synthesis followed by PCR amplification. Usually, each member of this DNA library has a T7 RNA polymerase transcription site and a ribosomal binding site at the 5’ end. The T7 promoter region allows large-scale in vitro T7 transcription to transcribe the DNA library into an mRNA library, which provides templates for the in vitro translation reaction later. The ribosomal binding site in the 5’-untranslated region (5’ UTR) is designed according to the in vitro translation system to be used. There are two popular commercially available in vitro translation systems. One is E. coli S30 Extract System (Promega) that requires a Shine-Dalgarno sequence in the 5’ UTR as a ribosomal binding site; [4] the other one is Red Nova Lysate (Novagen), which needs a ΔTMV ribosomal binding site.

Once the mRNA library is generated, it will be Urea-PAGE purified and ligated using T4 DNA ligase to the DNA spacer linker containing puromycin at the 3’ end. In this ligation step, a piece of mRNA is ligated with a single stranded DNA with the help from T4 DNA ligase. This is not a standard T4 DNA ligase ligation reaction, where two pieces of double stranded DNA are ligated together. To increase the yield of this special ligation, a single stranded DNA splint may be used to aid the ligation reaction. The 5’ terminus of the splint is designed to be complementary to the 3’ end of the mRNA, and the 3’ terminus of the splint is designed to be complementary to the 5’ end of the DNA spacer linker, which usually consists of poly dA nucleotides (Figure 2).

Figure 3. Selection Cycle. Figure 3. Selection Cycle.png
Figure 3. Selection Cycle.

The ligated mRNA-DNA-puromycin library is translated in Red Nova Lysate (Novagen) or E. coli S30 Extract System (Promega), resulting in polypeptides covalently linked in cis to the encoding mRNA. The in vitro translation can also be done in a PURE (protein synthesis using recombinant elements) system. PURE system is an E. coli cell-free translation system in which only essential translation components are present. Some components, such as amino acids and aminoacyl-tRNA synthases (AARSs) can be omitted from the system. Instead, chemically acylated tRNA can be added into the PURE system. It has been shown that some unnatural amino acids, such as N-methyl-amino acid accylated tRNA can be incorporated into peptides or mRNA-polypeptide fusions in a PURE system. [5]

After translation, the single-stranded mRNA portions of the fusions will be converted to heteroduplex of RNA/DNA by reverse transcriptase to eliminate any unwanted RNA secondary structures, and render the nucleic acid portion of the fusion more stable. This step is a standard reverse transcription reaction. For instance, it can be done by using Superscript II (GIBCO-BRL) following the manufacturer’s protocol.

The mRNA/DNA-polypeptide fusions can be selected over immobilized selection targets for several rounds (Figure 3). There might be a relatively high background for the first few rounds of selection, and this can be minimized by increasing selection stringency, such as adjusting salt concentration, amount of detergent, and/or temperature during the target/fusion binding period. Following binding selection, those library members that stay bound to the immobilized target are PCR amplified. The PCR amplification step will enrich the population from the mRNA-display library that has higher affinity for the immobilized target. Error-prone PCR can also be done in between each round of selection to further increase the diversity of the mRNA-display library and reduce background in selection. [6]

A less time-consuming protocol for mRNA display was recently published. [7]

Advantages

Although there are many other molecular display technologies, such as phage display, bacterial display, yeast display, and ribosome display, mRNA display technology has many advantages over the others. [8] The first three biological display libraries listed have polypeptides or proteins expressed on the respective microorganism’s surface and the accompanying coding information for each polypeptide or protein is retrievable from the microorganism’s genome. However, the library size for these three in vivo display systems is limited by the transformation efficiency of each organism. For example, the library size for phage and bacterial display is limited to 1-10 × 10^9 different members. The library size for yeast display is even smaller. Moreover, these cell-based display system only allow the screening and enrichment of peptides/proteins containing natural amino acids. In contrast, mRNA display and ribosome display are in vitro selection methods. They allow a library size as large as 10^15 different members. The large library size increases the probability to select very rare sequences, and also improves the diversity of the selected sequences. In addition, in vitro selection methods remove unwanted selection pressure, such as poor protein expression, and rapid protein degradation, which may reduce the diversity of the selected sequences. Finally, in vitro selection methods allow the application of in vitro mutagenesis [9] and recombination techniques throughout the selection process.

Although both ribosome display and mRNA display are in vitro selection methods, mRNA display has some advantage over the ribosome display technology. [10] mRNA display utilizes covalent mRNA-polypeptide complexes linked through puromycin; whereas, ribosome display utilizes stalled, noncovalent ribosome-mRNA-polypeptide complexes. [11] For ribosome display, selection stringency is limited to keep ribosome-mRNA-polypeptide in a complex because of the noncovalent ribosome-mRNA-polypeptide complexes. This may cause difficulties in reducing background binding during the selection cycle. Also, the polypeptides under selection in a ribosome display system are attached to an enormous rRNA-protein complex, a ribosome, which has a molecular weight of more than 2,000,000 Da. There might be some unpredictable interaction between the selection target and the ribosome, and this may lead to a loss of potential binders during the selection cycle. In contrast, the puromycin DNA spacer linker used in mRNA display technology is much smaller comparing to a ribosome. This linker may have less chance to interact with an immobilized selection target. Thus, mRNA display technology is more likely to give less biased results.

Application

In 1997, Roberts and Szostak showed that fusions between a synthetic mRNA and its encoded myc epitope could be enriched from a pool of random sequence mRNA-polypeptide fusions by immunoprecipitation. [6]

Nine years later, Fukuda and colleagues chose mRNA display method for in vitro evolution of single-chain Fv (scFv) antibody fragments. [12] They selected six different scFv mutants with five consensus mutations. However, kinetic analysis of these mutants showed that their antigen-specificity remained similar to that of the wild type. However, they have demonstrated that two of the five consensus mutations were within the complementarity determining regions (CDRs). And they concluded that mRNA display has the potential for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.

Roberts and coworkers have demonstrated that unnatural peptide oligomers consisting of an N-substituted amino acid can be synthesized as mRNA-polypeptide fusions. [13] N-substituted amino acid-containing peptides have been associated with good proteolytic stability and improved pharmacokinetic properties. This work indicates that mRNA display technology has the potential for selecting drug-like peptides for therapeutic usage resistant to proteolysis. [14]

See also

Related Research Articles

<span class="mw-page-title-main">Protein biosynthesis</span> Assembly of proteins inside biological cells

Protein biosynthesis is a core biological process, occurring inside cells, balancing the loss of cellular proteins through the production of new proteins. Proteins perform a number of critical functions as enzymes, structural proteins or hormones. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences.

Protein engineering is the process of developing useful or valuable proteins through the design and production of unnatural polypeptides, often by altering amino acid sequences found in nature. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It has been used to improve the function of many enzymes for industrial catalysis. It is also a product and services market, with an estimated value of $168 billion by 2017.

<span class="mw-page-title-main">Translation (biology)</span> Cellular process of protein synthesis

In biology, translation is the process in living cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids. This sequence is determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in addition of one specific amino acid to the protein being generated. The matching from nucleotide triple to amino acid is called the genetic code. The translation is performed by a large complex of functional RNA and proteins called ribosomes. The entire process is called gene expression.

A signal peptide is a short peptide present at the N-terminus of most newly synthesized proteins that are destined toward the secretory pathway. These proteins include those that reside either inside certain organelles, secreted from the cell, or inserted into most cellular membranes. Although most type I membrane-bound proteins have signal peptides, the majority of type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their first transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved. They are a kind of target peptide.

<span class="mw-page-title-main">Phage display</span> Biological technique to evolve proteins using bacteriophages

Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. These displaying phages can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.

Ribosome display is a technique used to perform in vitro protein evolution to create proteins that can bind to a desired ligand. The process results in translated proteins that are associated with their mRNA progenitor which is used, as a complex, to bind to an immobilized ligand in a selection step. The mRNA-protein hybrids that bind well are then reverse transcribed to cDNA and their sequence amplified via PCR. The result is a nucleotide sequence that can be used to create tightly binding proteins.

<span class="mw-page-title-main">Aptamer</span> Oligonucleotide or peptide molecules that bind specific targets

Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities, with variable levels of off-target binding and are sometimes classified as chemical antibodies. Aptamers and antibodies can be used in many of the same applications, but the nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. This difference can make aptamers a better choice than antibodies for some purposes.

The peptidyl transferase is an aminoacyltransferase as well as the primary enzymatic function of the ribosome, which forms peptide bonds between adjacent amino acids using tRNAs during the translation process of protein biosynthesis. The substrates for the peptidyl transferase reaction are two tRNA molecules, one bearing the growing peptide chain and the other bearing the amino acid that will be added to the chain. The peptidyl chain and the amino acids are attached to their respective tRNAs via ester bonds to the O atom at the CCA-3' ends of these tRNAs. Peptidyl transferase is an enzyme that catalyzes the addition of an amino acid residue in order to grow the polypeptide chain in protein synthesis. It is located in the large ribosomal subunit, where it catalyzes the peptide bond formation. It is composed entirely of RNA. The alignment between the CCA ends of the ribosome-bound peptidyl tRNA and aminoacyl tRNA in the peptidyl transferase center contribute to its ability to catalyze these reactions. This reaction occurs via nucleophilic displacement. The amino group of the aminoacyl tRNA attacks the terminal carboxyl group of the peptidyl tRNA. Peptidyl transferase activity is carried out by the ribosome. Peptidyl transferase activity is not mediated by any ribosomal proteins but by ribosomal RNA (rRNA), a ribozyme. Ribozymes are the only enzymes which are not made up of proteins, but ribonucleotides. All other enzymes are made up of proteins. This RNA relic is the most significant piece of evidence supporting the RNA World hypothesis.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

<span class="mw-page-title-main">EF-Tu</span> Prokaryotic elongation factor

EF-Tu is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes. It is found in eukaryotic mitochondria as TUFM.

Protein metabolism denotes the various biochemical processes responsible for the synthesis of proteins and amino acids (anabolism), and the breakdown of proteins by catabolism.

<span class="mw-page-title-main">Systematic evolution of ligands by exponential enrichment</span> Technique for producing oligonucleotides that specifically bind to a target

Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers. Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB and CASTing SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.

<span class="mw-page-title-main">Prokaryotic small ribosomal subunit</span> Smaller subunit of the 70S ribosome found in prokaryote cells

The prokaryotic small ribosomal subunit, or 30S subunit, is the smaller subunit of the 70S ribosome found in prokaryotes. It is a complex of the 16S ribosomal RNA (rRNA) and 19 proteins. This complex is implicated in the binding of transfer RNA to messenger RNA (mRNA). The small subunit is responsible for the binding and the reading of the mRNA during translation. The small subunit, both the rRNA and its proteins, complexes with the large 50S subunit to form the 70S prokaryotic ribosome in prokaryotic cells. This 70S ribosome is then used to translate mRNA into proteins.

In vitro compartmentalization (IVC) is an emulsion-based technology that generates cell-like compartments in vitro. These compartments are designed such that each contains no more than one gene. When the gene is transcribed and/or translated, its products become 'trapped' with the encoding gene inside the compartment. By coupling the genotype (DNA) and phenotype, compartmentalization allows the selection and evolution of phenotype.

<span class="mw-page-title-main">Expanded genetic code</span> Modified genetic code

An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids.

<span class="mw-page-title-main">Protein synthesis inhibitor</span> Inhibitors of translation

A protein synthesis inhibitor is a compound that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins.

The Streptavidin-Binding Peptide (SBP)-Tag is a 38-amino acid sequence that may be engineered into recombinant proteins. Recombinant proteins containing the SBP-Tag bind to streptavidin and this property may be utilized in specific purification, detection or immobilization strategies.

<span class="mw-page-title-main">Chloroplast DNA</span> DNA located in cellular organelles called chloroplasts

Chloroplast DNA (cpDNA) is the DNA located in chloroplasts, which are photosynthetic organelles located within the cells of some eukaryotic organisms. Chloroplasts, like other types of plastid, contain a genome separate from that in the cell nucleus. The existence of chloroplast DNA was identified biochemically in 1959, and confirmed by electron microscopy in 1962. The discoveries that the chloroplast contains ribosomes and performs protein synthesis revealed that the chloroplast is genetically semi-autonomous. The first complete chloroplast genome sequences were published in 1986, Nicotiana tabacum (tobacco) by Sugiura and colleagues and Marchantia polymorpha (liverwort) by Ozeki et al. Since then, a great number of chloroplast DNAs from various species have been sequenced.

<span class="mw-page-title-main">Affitin</span>

Affitins are artificial proteins with the ability to selectively bind antigens. They are structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaeal domain. By randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various targets, such as peptides, proteins, viruses, and bacteria.

<span class="mw-page-title-main">Translatomics</span>

Translatomics is the study of all open reading frames (ORFs) that are being actively translated in a cell or organism. This collection of ORFs is called the translatome. Characterizing a cell's translatome can give insight into the array of biological pathways that are active in the cell. According to the central dogma of molecular biology, the DNA in a cell is transcribed to produce RNA, which is then translated to produce a protein. Thousands of proteins are encoded in an organism's genome, and the proteins present in a cell cooperatively carry out many functions to support the life of the cell. Under various conditions, such as during stress or specific timepoints in development, the cell may require different biological pathways to be active, and therefore require a different collection of proteins. Depending on intrinsic and environmental conditions, the collection of proteins being made at one time varies. Translatomic techniques can be used to take a "snapshot" of this collection of actively translating ORFs, which can give information about which biological pathways the cell is activating under the present conditions.

References

  1. ↑ Roberts RW, Szostak JW (1997). "RNA-peptide fusions for the in vitro selection of peptides and proteins". PNAS . 94 (23): 12297–12302. Bibcode:1997PNAS...9412297R. doi: 10.1073/pnas.94.23.12297 . PMC   24913 . PMID   9356443.
  2. ↑ Liu R, Barrick JE, Szostak JW, Roberts RW (2000). "Optimized synthesis of RNA-protein fusions for in vitro protein selection". RNA-Ligand Interactions Part B. Methods in Enzymology. Vol. 318. pp. 268–93. doi:10.1016/S0076-6879(00)18058-9. ISBN   9780121822194. PMID   10889994.
  3. ↑ Kurz M, Gu K, Lohse PA (2000). "Psoralen photo-crosslinked mRNA–puromycin conjugates: a novel template for the rapid and facile preparation of mRNA–protein fusions". Nucleic Acids Research . 28 (18): 83e–83. doi:10.1093/nar/28.18.e83. PMC   110755 . PMID   10982894.
  4. ↑ Mattheakis LC, Bhatt RR, Dower WJ (1994). "An in vitro polysome display system for identifying ligands from very large peptide libraries". Proc Natl Acad Sci USA . 91 (19): 9022–6. Bibcode:1994PNAS...91.9022M. doi: 10.1073/pnas.91.19.9022 . PMC   44739 . PMID   7522328.
  5. ↑ Kawakami T, Murakami H, Suga H (January 2008). "Messenger RNA-programmed incorporation of multiple N-methyl-amino acids into linear and cyclic peptides". Chemistry & Biology . 15 (1): 32–42. doi:10.1016/j.chembiol.2007.12.008. PMID   18215771.
  6. 1 2 Roberts RW, Szostak JW (1997). "RNA-peptide fusions for the in vitro selection of peptides and proteins". Proc Natl Acad Sci USA . 94 (23): 12297–302. Bibcode:1997PNAS...9412297R. doi: 10.1073/pnas.94.23.12297 . PMC   24913 . PMID   9356443.
  7. ↑ Barendt PA, Ng DT, McQuade CN, Sarkar CA (2013). "Streamlined Protocol for mRNA Display". ACS Combinatorial Science . 15 (2): 77–81. doi:10.1021/co300135r. PMC   3666848 . PMID   23305392.
  8. ↑ Roberts RW (June 1999). "Totally in vitro protein selection using mRNA-protein fusions and ribosome display". Current Opinion in Chemical Biology . 3 (3): 268–73. doi:10.1016/S1367-5931(99)80042-8. PMID   10359713.
  9. ↑ Jing D, Li F, Jiang M, Cai J, Wu Y, Xie K, Wu X, Tang C, Liu J, Guo W, Shen G, Luo E (November 2013). "Pulsed Electromagnetic Fields Improve Bone Microstructure And Strength In Ovariectomized Rats". PLoS ONE . 8 (11): e79377. Bibcode:2013PLoSO...879377J. doi: 10.1371/journal.pone.0079377 . PMC   3828367 . PMID   24244491.
  10. ↑ Gold L (April 2001). "mRNA display: diversity matters during in vitro selection". Proc Natl Acad Sci USA . 98 (9): 4825–6. Bibcode:2001PNAS...98.4825G. doi: 10.1073/pnas.091101698 . PMC   33119 . PMID   11320229.
  11. ↑ Andrew Buchanan; Lutz Jermutus. "Ribosome Display or Mrna Display Method With Selection for Increased Stability of the Protein". Google Patents . Retrieved 9 June 2014.
  12. ↑ Fukuda I, Kojoh K, Tabata N, et al. (2006). "In vitro evolution of single-chain antibodies using mRNA display". Nucleic Acids Research . 34 (19): e127. doi:10.1093/nar/gkl618. PMC   1636464 . PMID   17012279.
  13. ↑ Frankel A, Millward SW, Roberts RW (November 2003). "Encodamers: unnatural peptide oligomers encoded in RNA" (PDF). Chemistry & Biology . 10 (11): 1043–50. doi: 10.1016/j.chembiol.2003.11.004 . PMID   14652071.
  14. ↑ White, E. Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M.; Danzig, Brittany A.; Hacker, David E.; Huie, Melissa; Williams, David C.; Edwards, Ross A. (2015-05-15). "Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C-Terminal Domain". ACS Chemical Biology . 10 (5): 1198–1208. doi:10.1021/cb500757u. PMC   4433557 . PMID   25654734.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.