Naoto Nemoto

Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 10¹³ species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential. To address this issue, we herein propose a novel DNA construction methodology based on the use of polymerase chain reaction (PCR) primers containing a single deoxyinosine (I) residue near their 5′ end. Treatment of the PCR products with endonuclease V generates 3′ overhangs with customized lengths and sequences, which can be ligated accurately and efficiently with other fragments having exactly complementary overhangs. As a proof of concept, we constructed an artificial gene library of single-domain antibodies from four DNA fragments.

Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems.

Peptides that recognize artificial materials including synthetic polymers and small molecules are drawing attention in the fields of biotechnology and chemical biology. In particular, reversible peptide aptamers that associate with the target molecules only under specific conditions are interesting. In this work, peptide aptamers that recognize a phenolphthalein derivative (PhP: a pH-sensitive organic dye) immobilized on a solid surface in a pH-dependent manner were selected using an in vitro display method (cDNA display). Considering the hydrophobic and aromatic nature of PhP, we prepared a biased DNA library (3A library) that encodes more aromatic amino acids than the standard random codon and performed seven rounds of selection from >10¹⁰ peptide species. The selected peptides including LVFLIWWM (LV59) associated with PhP-modified solid support (sepharose resin and magnetic beads) in neutral buffer but readily dissociated under basic conditions where PhP undergoes large structural change from lactone to quinoid, which is accompanied by increase of hydrophilicity and anionic charge. Control experiments suggested that LV59 recognized both phenol and lactone moieties, and the association under neutral pH is mainly driven by π-stacking and hydrophobic interaction between the peptide and PhP. Notably, however, total hydrophobicity and number of aromatic rings did not completely explain the affinity, and sequence specificity was observed to some extent. After further optimization, this interaction pair would be practically useful for protein purification.

Single-domain antibody (e.g., Nanobody, VHH antibody) is a promising scaffold for therapeutic and diagnostic reagents. To expand the range of target molecules, in vitro selection using cell-free display technologies such as cDNA display is useful and powerful because of their huge libraries and robust stability. We provide technical details for in vitro selection of single-domain antibodies using cDNA display.

The first in vitro selection of binding peptides against artificial lipid membranes from a random peptide library using an in vitro display method (cDNA display) is reported. The selected peptide, LB-1, has both amphiphilic and cationic regions, and proteins fused to LB-1 can be immobilized on the liposome surface.

In vitro display technologies such as mRNA and cDNA display are powerful tools to create and select functional peptides. However, in some cases, efficiency of mRNA-protein fusion is very low, which results in decreased library size and lower chance of successful selection. In this study, to improve mRNA-protein fusion efficiency, we prepared an mRNA display library of a protein with random N- and C-terminal coding regions consisting of 12 nucleotides (i.e. four amino acids), and performed an electrophoresis mobility shift assay (EMSA)-based selection of successfully formed mRNA display molecules. A single-domain antibody (Nanobody, or VHH) was used as a model protein, and as a result, a pair of sequences was identified that increased mRNA-protein fusion efficiency of this protein by approximately 20%. Interestingly, enhancement of the fusion efficiency induced by the identified sequences was protein-specific, and different results were obtained for other proteins including VHHs with different CDRs. The results suggested that conformation of mRNA as a whole, rather than the amino acid sequence of the translated peptide, is an important factor to determine mRNA-protein fusion efficiency.

RNA–protein interactions have a central role in the living world. In this article, we examined whether primitive peptides (30 residues) consisting of four types of amino acid (Gly, Ala, Asp, and Val) could interact with tRNA as a model of primitive RNAs in the RNA world. By in vitro selection of binding peptides using the cDNA display method, a characteristic peptide was selected from a random peptide library and assayed by electrophoretic mobility shift and pull-down assays. Interestingly, the selected peptide bound to a single-stranded region including a loop structure of an RNA molecule with some sequence specificity.