Shingo Ueno

In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

G protein-coupled receptors (GPCRs) are major drug targets, and their ligands are currently being explored and developed by many pharmaceutical companies and independent researchers. Class A (rhodopsin-like) GPCRs compose a predominant GPCR family; therefore, class A GPCR ligands are in demand. Growth hormone secretagogue receptor (GHS-R) is a class A GPCR that stimulates food intake by binding to its peptide ligand, ghrelin. Therefore, antagonists of GHS-R are expected to exert antiobesity function. In this article, we describe the use of cDNA display to screen for successfully and identify an antagonistic peptide of GHS-R. The antagonistic peptide inhibited the ghrelin-induced increase in intracellular Ca2+ in vitro (IC50 = approximately 10 μM) and repressed the contraction of isolated animal stomach in response to ghrelin. Furthermore, peripheral administration of the peptide inhibited the food intake of mice. This work provides new insight into the development of antiobesity drugs and describes a method for the discovery of unique peptide ligands for class A GPCRs.

cDNA Display: Rapid Stabilization of mRNA Display

The cDNA display method is a robust in vitro display technology that converts an unstable mRNA-protein fusion (mRNA display) to a stable mRNA/cDNA-protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein using a well-designed puromycin linker. We provide technical details for preparing cDNA display molecules and for the synthesis of the puromycin linker for the purpose of screening the functional proteins and peptides.

Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method

cDNA display using a puromycin-linker to covalently bridge a protein and its coding cDNA is a stable and efficient in vitro protein selection method. The optimal design of the often-used puromycin-linker is vital for effective selection. In this report, an improved puromycin-linker containing deoxyinosine bases as cleavage sites, which are recognized by endonuclease V, was introduced to extend the variety of the selection targets to molecules such as RNA. The introduction of this linker enables efficient in vitro protein selection without contamination from RNase T1, which is used for the conventional linker containing ribonucleotide G bases. In addition, mRNA-protein fusion efficiency was found to not depend on the length of the flexible poly (ethylene glycol) (PEG) region of the linker. These findings will allow practical and easy-to-use in vitro protein selection by cDNA display.

Microintaglio Printing of In situ Synthesized Proteins Enables Rapid Printing of High-Density Protein Microarrays Directly from DNA Microarrays

A simple and versatile approach to the simultaneous on-chip synthesis and printing of proteins has been studied for high-density protein microarray applications. The method used is based on the principle of intaglio printing using microengraved plates. Unlike conventional approaches that require multistep reactions for synthesizing proteins off the chip followed by printing using a robotic spotter, our approach demonstrates the following: (i) parallel and spotter-free printing of high-density protein microarrays directly from a type of DNA microarray and (ii) microcompartmentalization of cell-free coupled transcription/translation reaction and direct transferring of picoliter protein solution per spot to pattern microarrays of 25–100 µm features.

Evaluation of poly(dimethylsiloxane) microreactors for pattern size miniaturization of microintaglio-printing-based protein microarray

We developed a soft lithography-based microintaglio printing method for fabricating robot-free high-density protein microarrays using a poly(dimethylsiloxane) (PDMS) microchamber array. This method provides a simple approach to simultaneously synthesize and capture functional protein microarrays (with 25?100 ?m resolution in this study) directly from DNA microarrays in situ on a chip without a spotter and therefore can be an extremely effective tool for the miniaturization of arrays. However, minimizing the scale, which increases the PDMS surface area to sample volume ratio, can alter arraying outcomes because of interfacial phenomena. In this study, we evaluated the suitability of PDMS for miniaturizing cell-free synthesis-based protein microarrays and showed that the amount of a green fluorescent protein synthesized in situ inside PDMS microchambers monotonically decreased with decreasing microchamber (pattern) size and that the protein could not be detected in microchambers with a diameter smaller than 25 ?m. The impact of absorption and/or adsorption on the PDMS surface on protein synthesis inside the microchamber was observed, which was reduced by coating techniques. The results obtained here clearly suggest that PDMS interfacial phenomena can be suppressed while obtaining the benefits of cell-free protein synthesis and microintaglio printing, as a step toward developing ultrahigh-density protein microarrays.

Temperature-controlled microintaglio printing for high-resolution micropatterning of RNA molecules

We have developed an advanced microintaglio printing method for fabricating fine and high-density micropatterns and applied it to the microarraying of RNA molecules. The microintaglio printing of RNA reported here is based on the hybridization of RNA with immobilized complementary DNA probes. The hybridization was controlled by switching the RNA conformation via the temperature, and an RNA microarray with a diameter of 1.5 µm and a density of 40,000 spots/mm(2) with high contrast was successfully fabricated. Specifically, no size effects were observed in the uniformity of patterned signals over a range of microarray feature sizes spanning one order of magnitude. Additionally, we have developed a microintaglio printing method for transcribed RNA microarrays on demand using DNA-immobilized magnetic beads. The beads were arrayed on wells fabricated on a printing mold and the wells were filled with in vitro transcription reagent and sealed with a DNA-immobilized glass substrate. Subsequently, RNA was in situ synthesized using the bead-immobilized DNA as a template and printed onto the substrate via hybridization. Since the microintaglio printing of RNA using DNA-immobilized beads enables the fabrication of a microarray of spots composed of multiple RNA sequences, it will be possible to screen or analyze RNA functions using an RNA microarray fabricated by temperature-controlled microintaglio printing (TC-µIP).

Simultaneous Synthesis and Biotinylation of Proteins Using Puromycin-Based Labeling Technology for Fabrication of Protein Array Chip

Protein arrays represent a class of devices that are of growing importance in the field of proteomics. These arrays enable screening of a large amount of proteins in a short time and at a lower cost. Here we present a method to fabricate protein array using biotin-conjugated puromycin to simultaneously synthesize and label proteins followed by immobilization onto streptavidin-functionalized surface based on the noncovalent biotin-streptavidin interaction. This method demonstrates the fabrication of protein array based on cell-free transcription/translation system using unmodified DNA as a starting genetic material. As a consequence, the procedure of protein arraying has been greatly simplified over the conventional approaches that require tedious and multi-step reactions. Further, an integrated approach of micro reactor array technology makes this method very simple and robust for achieving high-density protein arrays.