Corrigendum: Possible roles for the hominoid-specific DSCR4 gene in human cells [Genes Genet. Syst. (2021) 96, p. 1-11].

Legends to Figures 4 and 5 (p. 7) should be exchanged. Below are the correct legends to Figure 4 and Figure 5. Fig. 4. Interconnection of DSCR4 overexpression-mediated perturbed pathways. KEGG analysis of DSCR4 overexpression-mediated DEGs shows enrichment for the tightly interconnected pathways of the coagulation cascade and the complement cascade (highlighted in red) and further confirm the connection of these cascades with cell adhesion, migration and proliferation (red circle). Fig. 5. Expression profile of DSCR4 across human cell lines and tissues. According to Roadmap Epigenomics Project data, DSCR4 and DSCR8, which share a bidirectional promoter, are highly expressed only in K562 cells, a type of leukemia cell. Analysis of transcriptome data provided by Prescott et al. (2015) showed that DSCR4 and DSCR8 also display high expression in human and chimpanzee neural crest cells, which are critical migratory cells involved in facial morphogenesis in the embryo. (1) Data from Prescott et al. (2015). (2) Samples also include esophagus, lung, spleen and fetal large intestine. (3) Samples also include brain germinal matrix, hippocampus, fetal small intestine, stomach, left ventricle, small intestine, sigmoid colon, HEPG2 cells and HMEC cells. The PDF file for DOI: https://doi.org/10.1266/ggs.20-00012 has been replaced with the corrected version as of June 17, 2021.

Selection of Ovalbumin-specific Binding Peptides through Instant Translation in Ribosome Display Using E. coli Extract.

In vitro selection has been widely used to generate molecular-recognition elements in analytical sciences. Although reconstituted types of in vitro transcription and translation (IVTT) system, such as PURE system, are nowadays widely used for ribosome display and mRNA/cDNA display, use of E. coli extract is often avoided, presumably because it contains unfavorable contaminants, such as ribonuclease. Nevertheless, the initial speed of protein translation in E. coli extract is markedly faster than that of PURE system. We thus hypothesized that E. coli extract is more appropriate for instant translation in ribosome display than PURE system. Here, we first revisit the potency of E. coli extract for ribosome display by shortening the translation time, and then applied the optimized condition for selecting peptide aptamers for ovalbumin (OVA). The OVA-binding peptides selected using E. coli extract exhibited specific binding to OVA, even in the presence of 50% serum. We conclude that instant translation in ribosome display using E. coli extract has the potential to generate easy-to-use and economical molecular-recognition elements in analytical sciences.

Phosphorogenic and spontaneous formation of tris(bipyridine)ruthenium in peptide scaffolds.

Redox-active ruthenium complexes have been widely used in various fields; however, the harsh conditions required for their synthesis are not always conducive to their subsequent use in biological applications. In this study, we demonstrate the spontaneous formation of a derivative of tris(bipyridine)ruthenium at 37°C through the coordination of three bipyridyl ligands incorporated into a peptide to a ruthenium ion. Specifically, we synthesized six bipyridyl-functionalized peptides with randomly chosen sequences. The six peptides bound to ruthenium ions and exhibited similar spectroscopic and electrochemical features to tris(bipyridine)ruthenium, indicating the formation of ruthenium complexes as we anticipated. The photo-excited triplet state of the ruthenium complex formed in the peptides exhibited an approximately 1.6-fold longer lifetime than that of tris(bipyridine)ruthenium. We also found that the photo-excited state of the ruthenium complexes was able to transfer an electron to methyl viologen, indicating that the ruthenium complexes formed in the peptides had the same ability to transfer charge as tris(bipyridine)ruthenium. We believe that this strategy of producing ruthenium complexes in peptides under mild conditions will pave the way for developing new metallopeptides and metalloproteins containing functional metal-complexes.

In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection.

An electrosensitive peptide probe has been developed from an in vitro selection technique using biorthogonal tRNA prepared with an electroreactive non-natural amino acid, 3,4-ethylenedioxythiophene-conjugated aminophenylalanine. The selected probe quantitatively detected the influenza virus based on a signal "turn-on" mechanism. The developed strategy could be used to develop electrochemical biosensors toward a variety of targets.

Wash-free and selective imaging of epithelial cell adhesion molecule (EpCAM) expressing cells with fluorogenic peptide ligands.

Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step to identify circulating tumor cells (CTCs) from blood. To detect the EpCAM, we here designed and synthesized a series of fluorogenic peptides. Specifically, we functionalized an EpCAM-binding peptide, Ep114, by replacing its amino acids to an aminophenylalanine that was modified with environmentally sensitive 7-nitro-2,1,3-benzoxadiazole (NBD-amPhe). Among six synthesized peptides, we have found that two peptides, Q4X and V6X (X represents NBD-amPhe), retain the Ep114's binding ability and specifically mark EpCAM-expressing cells by just adding these peptides to the cultivation medium. Our wash-free, fluorogenic peptide ligands would boost the development of next generation devices for CTC diagnoses.

"All-in-one" in vitro selection of collagen-binding vascular endothelial growth factor.

To enhance the therapeutic effect of growth factors, a powerful strategy is to direct their localization to damaged sites. To treat skin wounds and myocardial infarction, we selected vascular endothelial growth factor (VEGF) carrying binding affinity to collagen. A simple conjugation of a reported collagen-binding sequence and VEGF did not increase the collagen-binding affinity, indicating that the molecular interaction between the two proteins abolished collagen binding activity. Here, we present a new molecular evolution strategy, "all-in-one" in vitro selection, in which a collagen-binding VEGF (CB-VEGF) was directly identified from a random library consisting of random and VEGF sequences. As expected, the selected CB-VEGFs exhibited high binding affinity to collagen and maintained the same growth enhancement activity for endothelial cells as unmodified VEGF in solution. Furthermore, the selected CB-VEGF enhanced angiogenesis at skin wounds and infarcted myocardium. This study demonstrates that "all-in-one" in vitro selection is a novel strategy for the design of functional proteins for regenerative medicine.

Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group.

To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity.

Two site genetic incorporation of varying length polyethylene glycol into the backbone of one peptide.

Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of a polypeptide using stop-anticodon and frameshift anticodon-containing tRNAs, which were acylated with PEG-containing amino acids.

In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads.

Photoresponsive peptide aptamer to glutathione-immobilized microbeads was in vitro selected using ribosome display incorporated with tRNA carrying an amino acid coupled with an azobenzene.

A fluorogenic peptide probe developed by in vitro selection using tRNA carrying a fluorogenic amino acid.

A peptide that binds and emits fluorescence in response to conformational change in a target protein was developed by in vitro selection using tRNA carrying a fluorogenic amino acid. This technology could prove to be useful for the development of separation-free immunoassays and bio-imaging analyses.

Wash-free, electrochemical platform for the quantitative, multiplexed detection of specific antibodies.

The diagnosis, prevention, and treatment of many illnesses, including infectious and autoimmune diseases, would benefit from the ability to measure specific antibodies directly at the point of care. Thus motivated, we designed a wash-free, electrochemical method for the rapid, quantitative detection of specific antibodies directly in undiluted, unprocessed blood serum. Our approach employs short, contiguous polypeptide epitopes coupled to the distal end of an electrode-bound nucleic acid "scaffold" modified with a reporting methylene blue. The binding of the relevant antibody to the epitope reduces the efficiency with which the redox reporter approaches, and thus exchanges electrons with, the underlying sensor electrode, producing readily measurable change in current. To demonstrate the versatility of the approach, we fabricated a set of six such sensors, each aimed at the detection of a different monoclonal antibody. All six sensors are sensitive (subnanomolar detection limits), rapid (equilibration time constants ∼8 min), and specific (no appreciable cross reactivity with the targets of the other five). When deployed in a millimeter-scale, an 18-pixel array with each of the six sensors in triplicate support the simultaneous measurement of the concentrations of multiple antibodies in a single, submilliliter sample volume. The described sensor platform thus appears be a relatively general approach to the rapid and specific quantification of antibodies in clinical materials.

Detection of telomerase activity in high concentration of cell lysates using primer-modified gold nanoparticles.

Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.

The rate of intramolecular loop formation in DNA and polypeptides: the absence of the diffusion-controlled limit and fractional power-law viscosity dependence.

The problem of determining the rate of end-to-end collisions for polymer chains has attracted the attention of theorists and experimentalists for more than three decades. The typical theoretical approach to this problem has focused on the case where a collision is defined as any instantaneous fluctuation that brings the chain ends to within a specific capture distance. In this paper, we study the more experimentally relevant case, where the end-to-end collision dynamics are probed by measuring the excited state lifetime of a fluorophore (or other lumiphore) attached to one chain end and quenched by a quencher group attached to the other end. Under this regime, a "contact" is defined not by the chain ends approach to within some sharp cutoff but, instead, typically by an exponentially distance-dependent process. Previous theoretical models predict that, if quenching is sufficiently rapid, a diffusion-controlled limit is attained, where such measurements report on the probe-independent, intrinsic end-to-end collision rate. In contrast, our theoretical considerations, simulations, and an analysis of experimental measurements of loop closure rates in single-stranded DNA molecules all indicate that no such limit exists, and that the measured effective collision rate has a nontrivial, fractional power-law dependence on both the intrinsic quenching rate of the fluorophore and the solvent viscosity. We propose a simple scaling formula describing the effective loop closure rate and its dependence on the viscosity, chain length, and properties of the probes. Previous theoretical results are limiting cases of this more general formula.

The length and viscosity dependence of end-to-end collision rates in single-stranded DNA.

Intramolecular dynamics play an essential role in the folding and function of biomolecules and, increasingly, in the operation of many biomimetic technologies. Thus motivated we have employed both experiment and simulation to characterize the end-to-end collision dynamics of unstructured, single-stranded DNAs ranging from 6 to 26 bases. We find that, because of the size and flexibility of the optical reporters employed experimentally, end-to-end collision dynamics exhibit little length dependence at length scales <11 bases. For longer constructs, however, the end-to-end collision rate exhibits a power-law relationship to polymer length with an exponent of -3.49 +/- 0.13. This represents a significantly stronger length dependence than observed experimentally for unstructured polypeptides or predicted by polymer scaling arguments. Simulations indicate, however, that the larger exponent stems from electrostatic effects that become important over the rather short length scale of these highly charged polymers. Finally, we have found that the end-to-end collision rate also depends linearly on solvent viscosity, with an experimentally significant, nonzero intercept (the extrapolated rate at zero viscosity) that is independent of chain length--n observation that sheds new light on the origins of the "internal friction" observed in the dynamics of many polymer systems.

Time-resolved small-angle X-ray scattering investigation of the folding dynamics of heme oxygenase: implication of the scaling relationship for the submillisecond intermediates of protein folding.

Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (Rg) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method. The folding process of HO was found to contain a transient oligomerization; however, the conformation within 10 ms was demonstrated to be monomeric and to possess Rg of 26.1(+/-1.1) A. Together with the corresponding data for proteins with different chain lengths, the seven Rg values demonstrated the scaling relationship to chain length with a scaling exponent of 0.35+/-0.11, which is close to the theoretical value of 1/3 predicted for globules in solutions where monomer-monomer interactions are favored over monomer-solvent interactions (poor solvent). The finding indicated that the initial folding dynamics of proteins bears the signature of the coil-globule transition, and offers a clue to explain the folding mechanisms of proteins with different chain lengths.

Specifically collapsed intermediate in the early stage of the folding of ribonuclease A.

Nature of the burst-phase signals of protein folding has been the subject of much debate as to whether the signals represent the formation of early intermediates or the non-specific collapse of unfolded polypeptides. To distinguish the two possibilities, the submillisecond folding dynamics of ribonuclease A (RNase A) was examined, and compared with those of the disulfide bond-ruptured analog of RNase A (r-RNase A). The circular dichroism measurements on RNase A showed the burst-phase signal within 320 micros after the initiation of the folding reaction, which was identical to that observed for r-RNase A. In contrast, the burst phase increase in the extrinsic fluorescence from 1-anilino-8-naphthalene sulfonate (ANS) was observed for RNase A but not for r-RNase A. The kinetic titration experiment of the ANS fluorescence intensity showed the presence of a specific binding site for ANS in the fast-refolding component of RNase A. The small-angle X-ray scattering measurements at approximately 22 ms after initiating the folding reaction demonstrated that the burst phase conformations of the medium and slow-refolding components of RNase A were distinctly smaller than that of r-RNase A. These results indicated the difference in the burst phase conformations of RNase A and r-RNase A. Since r-RNase A is denatured in the physiological solution condition, the burst-phase signal of RNase A was interpreted as the formation of the folding intermediate with specific conformations.