Short lifetime structures appearing in RNA and DNA.

The structure and function of unstable single-stranded DNA (ssDNA) have not been widely examined. While numerous studies have investigated DNA as an information molecule, the different potentials of DNA, particularly those of ssDNA, remain unclear. For polypeptides, the significance of denatured structures has been established in the past two decades. Polynucleotides have chemically distinct properties from polypeptides, but their behaviours have not been thoroughly studied. In this review, three different phenomena related to unstable ssDNA are discussed: i) ssDNA cleavage of restriction enzymes; ii) single-stranded conformation polymorphism, which can be theoretically explained by single-stranded conformation dynamics; and iii) random PCR (Polymerase Chain Reaction). These features can be utilized for scientific or technical applications. Previous studies showed that the phenomena exhibited by ssDNA were correctly understood only when unstable and transient structures were taken into account. Transient structures of ssDNA may have undiscovered functions governed by very rapid processes and/or multi-diversity states because of their intrinsic natures.

'Head-to-Head' mRNA display for the translation of multi-copied proteins with a free C-terminus.

With the development of various methods for affinity-based selection of proteins such as phage display, ribosomal display, and mRNA display, the progress in this field has been gradually shifting to function-based selection, such as through single-molecule observation, genetic selection, and compartmentalization technologies. In this vein, we present an opposite link mode of mRNA display termed as a 'Head-to-Head' (H2H) link. The key technique in H2H, formation of a covalent bond between O6-benzylguanine (BG) and O6-alkylguanine-DNA alkyltransferase (AGT), was demonstrated to be workable in H2H ligation, where mRNA is linked to a nascent AGT via a BG-DNA linker, resulting in a "(C-terminus) protein-BG-DNA linker-mRNA (5'-terminus)" conjugate. Thus, a head (N-terminus) to head (5'-terminus) linkage is formed. Among the advantages of H2H, the generation of multi-copied proteins is the most promising and was proven to be possible owing to the restored stop codon, which had been intentionally removed in the conventional mRNA display. Another advantage is obviously having a free C-terminus of the protein, which can be used for modifications such as C-terminal methylation, α-amidation, and others, which occur in nature. A superior merit of H2H is that it makes it possible to use a single construct commonly in mRNA display (affinity-based) and compartmentalization technologies (function-based) without requiring complicated construct changes.

Peptide aptamer-modified single-walled carbon nanotube-based transistors for high-performance biosensors.

Biosensors employing single-walled carbon nanotube field-effect transistors (SWCNT FETs) offer ultimate sensitivity. However, besides the sensitivity, a high selectivity is critically important to distinguish the true signal from interference signals in a non-controlled environment. This work presents the first demonstration of the successful integration of a novel peptide aptamer with a liquid-gated SWCNT FET to achieve highly sensitive and specific detection of Cathepsin E (CatE), a useful prognostic biomarker for cancer diagnosis. Novel peptide aptamers that specifically recognize CatE are engineered by systemic in vitro evolution. The SWCNTs were firstly grown using the thermal chemical vapor deposition (CVD) method and then were employed as a channel to fabricate a SWCNT FET device. Next, the SWCNTs were functionalized by noncovalent immobilization of the peptide aptamer using 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker. The resulting FET sensors exhibited a high selectivity (no response to bovine serum albumin and cathepsin K) and label-free detection of CatE at unprecedentedly low concentrations in both phosphate-buffered saline (2.3 pM) and human serum (0.23 nM). Our results highlight the use of peptide aptamer-modified SWCNT FET sensors as a promising platform for near-patient testing and point-of-care testing applications.

DNA-based mutation assay GPMA (genome profiling-based mutation assay): reproducibility, parts-per-billion scale sensitivity, and introduction of a mammalian-cell-based approach.

Genome profiling-based mutation assay (GPMA) is, to date, the only DNA sequence-based mutation assay that directly measures DNA alterations induced by mutagens. Here, the all-important congruence of mutagen assignment between DNA-based GPMA and the phenotype-based Ames test (the gold standard of mutagen assays) was confirmed qualitatively and semi-quantitatively by means of 94 chemical species (including previously examined 64). The high sensitivity (on the order of 10 ppb) and reproducibility of GPMA were also corroborated by the match between virtually independent experiments conducted in the distant past (10 years ago) and recently. Meanwhile, a standard experimental framework was established: the conditions of 100 parts per billion (ppb) concentration of a chemical and 15-generation culture of Escherichia coli. Moreover, a mammalian cell line (NIH 3T3) was shown to be suitable as a tester organism for the GPMA approach. Preliminary experimental results suggested that this approach can provide a qualitatively equivalent and quantitatively different mutagen assay results relative to the bacteria-based GPMA (renamed as bGPMA). This finding confirmed the effectiveness of the GPMA approach and indicates that mGPMA is a promising way to detect mammalian-cell mutagens.

Ordered genome change of plant and animal body cells revealed by the genome profiling method.

In the past, it was widely thought that, although epigenetically different, the genome sequences of cells are basically the same in a single body. In retrospect, the genome-uniformity idea may have been naïve, considering that DNA polymerases cannot be perfect. Here, a systemic, not sporadic, genome change was demonstrated in a single plant (Arabidopsis) and animal (zebrafish) body using genome DNAs taken in an ordered manner using the genome profiling method. This can be explained because mutations accumulate additively in progeny cells, and these results are critically significant for developmental and oncological research.

Expression of moloney murine leukemia virus reverse transcriptase in a cell-free protein expression system.

OBJECTIVE: To characterize Moloney murine leukemia virus (MMLV) reverse transcriptases (RTs) expressed in a cell-free system and in Escherichia coli. RESULTS: We previously expressed MMLV RT using an E. coli expression system and generated a highly thermostable quadruple variant MM4 (E286R/E302K/L435R/D524A) by site-directed mutagenesis. In this study, we expressed the wild-type MMLV RT (WT) and MM4 using a cell-free protein expression system from insect cells. WT exhibited DNA polymerase and RNase H activities, while MM4, in which the catalytic residue for RNase H activity, Asp524 is changed into Ala, exhibited only DNA polymerase activity. MM4, when held at 60 °C for 10 min, retained DNA polymerase activity, while WT, held at 54 °C for 10 min, lost this activity. In the cDNA synthesis reaction (0.5 µl) in which WT or MM4 were exposed to various temperatures and amounts of target RNA in a microarray chip, MM4 exhibited higher thermostability than WT. CONCLUSION: MMLV RT expressed in the cell-free system is indistinguishable from that expressed in E. coli.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.

Strong inhibition of beta-amyloid peptide aggregation realized by two-steps evolved peptides.

Several decades of cumulated research evidence have proven that aggregation of beta-amyloid 42 (Aß42) is the main cause of neuronal death in the brains of patients with Alzheimer's disease. Therefore, inhibition of Aß42 aggregation holds great promise for the prevention and treatment of Alzheimer's disease. To this end, we used a systematic in vitro evolution including a paired peptide library method. We identified two peptides with high binding affinity (with Kd in the nm range) for Aß42. Functionally, these peptides strongly inhibited the aggregation of Aß42 as shown by the thioflavin T assay and atomic force microscopy. Moreover, these peptides rescued PC12 cells from the cytotoxic effect of aggregated Aß42 in vitro. Our results suggest that these novel peptides may be potential therapeutic seeds for the treatment of Alzheimer's disease.

A radioisotope-nondependent high-sensitivity method for measuring the activity of glioblastoma-related O(6)-methylguanine DNA methyltransferase.

O(6)-Methylguanine DNA methyltransferase (MGMT) cancels the anticancer effect of temozolomide (drug for glioblastoma), which introduces methylation to DNA. Therefore, developing an MGMT inhibitor is a promising strategy for the treatment of this cancer. For this purpose, a sensitive detection method that does not depend on the conventional radioisotope (RI) method was developed. This was realized by a fluorescence-based method that measured the amount of cleavable restriction sites demethylated by the action of MGMT; this method was enhanced by introducing a polymerase chain reaction (PCR) amplification step. As an assay of enzyme activity, 20-fold higher sensitivity (subnanomolar) was attained compared with our and others' fluorescence-based approaches.

Amino group binding peptide aptamers with double disulphide-bridged loops selected by in vitro selection using cDNA display.

Peptide aptamers that specifically bind to the amino group on a solid-phase were screened by in vitro selection using the cDNA display method. The identified peptides have a unique structure containing two cyclic loops with disulphide bonds and a linkage region, which were indispensable for molecular recognition.

Systematic genome sequence differences among leaf cells within individual trees.

BACKGROUND: Even in the age of next-generation sequencing (NGS), it has been unclear whether or not cells within a single organism have systematically distinctive genomes. Resolving this question, one of the most basic biological problems associated with DNA mutation rates, can assist efforts to elucidate essential mechanisms of cancer. RESULTS: Using genome profiling (GP), we detected considerable systematic variation in genome sequences among cells in individual woody plants. The degree of genome sequence difference (genomic distance) varied systematically from the bottom to the top of the plant, such that the greatest divergence was observed between leaf genomes from uppermost branches and the remainder of the tree. This systematic variation was observed within both Yoshino cherry and Japanese beech trees. CONCLUSIONS: As measured by GP, the genomic distance between two cells within an individual organism was non-negligible, and was correlated with physical distance (i.e., branch-to-branch distance). This phenomenon was assumed to be the result of accumulation of mutations from each cell division, implying that the degree of divergence is proportional to the number of generations separating the two cells.

Repression of cathepsin E expression increases the risk of mammary carcinogenesis and links to poor prognosis in breast cancer.

Despite advances in detection and treatment for breast cancer (BC), recurrence and death rates remain unacceptably high. Therefore, more convenient diagnostic and prognostic methods still required to optimize treatments among the patients. Here, we report the clinical significance of the serum cathepsin E (CatE) activity as a novel prognostic marker for BC. Correlation analysis between the serum levels of CatE expression and clinicopathological parameters revealed that the activity levels, but not the protein levels, were negatively associated with the stages and progression of BC. Univariate and multivariate analyses demonstrated that the serum CatE activity was significantly correlated with favorable prognostic outcomes of the patients. The functional link of CatE expression to BC progression was further corroborated by in vivo and in vitro studies with mice exhibiting different levels of CatE expression. Multiparous CatE (-) (/) (-) mice spontaneously developed mammary tumors concomitant with morphological transformation and altered growth characteristics of the mammary glands. These alterations were associated in part with the induction of epithelial-mesenchymal transition and the activation of ß-catenin-dependent pathway in mammary cells. Loss of CatE strongly induced the translocation and accumulation of Wnt5a in the nuclei, thereby leading to the aberrant trafficking, maturation and secretion of Wnt5a and the impaired signaling. The interaction of CatE and Wnt5a was verified by proximity ligation assay and by knockdown or restoration of CatE expression in the mammary cells. Consequently, our data demonstrate that CatE contributes to normal growth and development of mammary glands through proper trafficking and secretion of Wnt5a.

Estimation of statistical binding properties of ligand population during in vitro selection based on population dynamics theory.

During in vitro selection process, it is very valuable to monitor the binding properties of the ligand population in real time, particularly the population average of the association constant in the population. If this monitoring can be realized, the selection process can be controlled in a rational way. In this paper, we present a simple method to estimate the binding properties of the ligand population during in vitro selection. The framework of the method is as follows. First, the number of all the collected ligand molecules, which are eluted after incubation and washing, is measured. Ideally, this number corresponds to the number of all the ligand molecules bound with the target-receptor or other materials in a test tube. This measurement is performed through several successive rounds of selection. Second, the measured numbers of molecules are subjected to a theoretical analysis, based on the mathematical theory of population dynamics in the selection process. Then, we can estimate the probability density of the binding free energy in the ligand population. The validity of our method was confirmed by several computer simulations based on a physicochemical model.

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 Ca(2+) in vitro (IC(50) = 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.

Peptide-Modulated Activity Enhancement of Acidic Protease Cathepsin E at Neutral pH.

Enzymes are regulated by their activation and inhibition. Enzyme activators can often be effective tools for scientific and medical purposes, although they are more difficult to obtain than inhibitors. Here, using the paired peptide method, we report on protease-cathepsin-E-activating peptides that are obtained at neutral pH. These selected peptides also underwent molecular evolution, after which their cathepsin E activation capability improved. Thus, the activators we obtained could enhance cathepsin-E-induced cancer cell apoptosis, which indicated their potential as cancer drug precursors.

Simple and tunable Förster resonance energy transfer-based bioprobes for high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry.

Sensing systems based on Förster resonance energy transfer (FRET) can be used to monitor enzymatic reactions, protein-protein interactions, changes in conformation, and Ca2+ oscillations in studies on cellular dynamics. We developed a series of FRET-based chimeric bioprobes, each consisting of fluorescent protein attached to a fluorescent dye. Green and red fluorescent proteins were used as donors and a series of Alexa Fluor dyes was used as acceptors. The basic fluorescent proteins were substituted with appropriate amino acids for recognition of the target (caspase-3) and subjected to site-directed modification with a fluorescent dye. Variants that retained similar emission profiles to the parent proteins were readily derived for use as FRET-based bioprobes with various fluorescent patterns by incorporating various fluorescent proteins and dyes, the nature of which could be adjusted to experimental requirements. All the constructs prepared functioned as bioprobes for quantitative measurement of caspase-3 activity in vitro. Introduction of the bioprobes into cells was so simple and efficient that activation of caspase-3 upon apoptosis could be monitored by means of cytometric analysis. FRET-based bioprobes are valuable tool for high-throughput flow-cytometric analysis of many cellular events when used in conjunction with other fluorescent labels or markers. Statistical dynamic studies on living cells could provide indications of paracrine signaling.

Trials of the detection of semen and vaginal fluid RNA using the genome profiling method.

The identification of sperm at the scene of a sexual crime is important evidence that can be used to prove that a crime took place. We used the new genome profiling (GP) method in this study to identify sperm and vaginal fluid from RNA extracted from bodily fluids. We randomly amplified genes via a PCR approach from these semen and vaginal fluid samples and performed temperature gradient gel electrophoresis between 15-65°C. We identified specific species identification dots (spiddos) for semen and vaginal fluid. The results showed that the GP method is effective for the identification of bodily fluids at the scene of a sex crime.

In Vitro Selection of Cathepsin E-Activity-Enhancing Peptide Aptamers at Neutral pH.

The aspartic protease cathepsin E has been shown to induce apoptosis in cancer cells under physiological conditions. Therefore, cathepsin E-activity-enhancing peptides functioning in the physiological pH range are valuable potential cancer therapeutic candidates. Here, we have used a general in vitro selection method (evolutionary rapid panning analysis system (eRAPANSY)), based on inverse substrate-function link (SF-link) selection to successfully identify cathepsin E-activity-enhancing peptide aptamers at neutral pH. A successive enrichment of peptide activators was attained in the course of selection. One such peptide activated cathepsin E up to 260%, had a high affinity (K(D); ∼300 nM), and had physiological activity as demonstrated by its apoptosis-inducing reaction in cancerous cells. This method is expected to be widely applicable for the identification of protease-activity-enhancing peptide aptamers.

Toward the fast blind docking of a peptide to a target protein by using a four-body statistical pseudo-potential.

In vitro molecular evolution creates a lot of peptide aptamers that bind to each target protein. In many cases, their binding sites on a protein surface are not known. Then, predicting the binding sites through computation within a reasonable time is desirable. With this aim, we have developed a novel system of fast and robust blind docking of a peptide to a fixed protein structure at low computational costs. Our algorithm is based on the following scheme. Representing each of the amino acid residues by a single point corresponding to its side-chain center, the structure of a target protein and that of a ligand peptide are coarse-grained. The peptide, which is described as a flexible bead model, is movable along the grid-points which are set surrounding the protein. An arbitrary state of the protein-peptide complex is subjected to Delaunay tessellation. Then, the fitness of a peptide-coordination to the protein is measured by a four-body statistical pseudo-potential. Through 1000 trials of a simple hill-climbing optimization, the best 15 peptide-coordinations with the 1st-15th highest fitness values are selected as candidates for the putative coordination. Retrieving the available 28 protein-peptide complexes from the Protein Databank, we carried out the blind docking test for each system. The best 15 peptide-coordinations fell into several clusters by the cluster analysis based on their spatial distribution. We found that, in most cases, the largest cluster or second largest cluster correspond to nearly correct binding sites, and that the mean (+/- standard deviation) of GTGD over all the 28 cases is 4.8 A(+/-3.8 A), where GTGD represents the distance from the putative binding site to the correct binding site.

High-throughput three-dimensional gel electrophoresis for versatile utilities: a stacked slice-gel system for separation and reactions (4SR).

A novel high-throughput system, called the stacked slice-gel system for separation and reactions (4SR), was developed for the analysis of DNA/RNA and protein/peptide. The system provides a novel three-dimensional gel electrophoresis approach that exploits the property of stacked slice gels. It allows multiple samples simultaneously to react as well as to be separated, offering a two-dimensional (mxn) sample loading system. For this purpose, high-throughput multi-micro vessels (MMVs) containing variable numbers of wells (100 wells in this paper) have been used, which are made of 25 mm square-size polyacrylamide gels. Furthermore, after electrophoretic separation, a slice gel containing a desired sample can be easily removed and proceeded to the next step. Different biological reactions as well as successive separation of products were effectively carried out dealing with DNA/RNA and protein/peptide. It shows that this system has a diversity of potentials to be developed.