An RNA Binding Peptide Consisting of Four Types of Amino Acid by in Vitro Selection Using cDNA Display.

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.

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.

Optimal terminal sequences for continuous or serial isothermal amplification of dsRNA with norovirus RNA replicase.

The norovirus RNA replicase (NV3D(pol), 56 kDa, single chain monomeric protein) can amplify double-stranded (ds) RNA isothermally. It will play an alternative role in the in vitro evolution against traditional Qß RNA replicase, which cannot amplify dsRNA and consists of four subunits, three of which are borrowed from host E.coli. In order to identify the optimal 3'-terminal sequence of the RNA template for NV3D(pol), an in vitro selection using the serial transfer was performed for a random library having the 3'-terminal sequence of ---UUUUUUNNNN-3'. The population landscape on the 4-dimensional sequence space of the 17(th) round of transfer gave a main peak around ---CAAC-3'. In the preceding studies on the batch amplification reaction starting from a single-stranded RNA, a template with 3'-terminal C-stretch was amplified effectively. It was confirmed that in the batch amplification the ---CCC-3' was much more effective than the ---CAAC-3', but in the serial transfer condition in which the ----CAAC-3' was sustained stably, the ---CCC-3' was washed out. Based on these results we proposed the existence of the "shuttle mode" replication of dsRNA. We also proposed the optimal terminal sequences of RNA for in vitro evolution with NV3D(pol).

Characterization of norovirus RNA replicase for in vitro amplification of RNA.

BACKGROUND: The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Qß replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3D(pol)) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3D(pol) in vitro were re-evaluated in this context. RESULTS: NV3D(pol), synthesized by using a cell-free translation system, represented the activities which were reported in the previous several studies and the reports were not fully consistent each other. The efficiency of the initiation of replication was dependent on the 3'-terminal structure of single-stranded RNA template, and especially, NV3D(pol) preferred a self-priming small stem-loop. In the non-self-priming and primer-independent replication reaction, the presence of -CCC residues at the 3'-terminus increased the initiation efficiency and we demonstrated the one-pot isothermal RNA (even dsRNA) amplification by 16-fold. NV3D(pol) also showed a weak activity of elongation-reaction from a long primer. Based on these results, we present a scheme of the primer-independent isothermal amplification of RNA with NV3D(pol) in vitro. CONCLUSIONS: NV3D(pol) can be used as an RNA replicase in in vitro RNA + protein evolution with the RNA of special terminal sequences.

Theoretical consideration of selective enrichment in in vitro selection: optimal concentration of target molecules.

We considered an in vitro selection system composed of a peptide-ligand library and a single target protein receptor, and examined effective strategies to realize maximum efficiency in selection. In the system, a ligand molecule with sequence s binds to a target receptor with probability of [R]/(K(ds)+[R]) (specific binding) or binds to non-target materials with probability of q (non-specific binding), where [R] and K(ds) represent the free target-receptor concentration at equilibrium and dissociation constant K(d) of the ligand sequence s with the receptor, respectively. Focusing on the fittest sequence with the highest affinity (represented by K(d1) ≡ min{K(ds)|s=1,2,…,M}) in the ligand library with a library size N and diversity M, we examined how the target concentration [R] should be set in each round to realize the maximum enrichment of the fittest sequence. In conclusion, when N >> M (that realizes a deterministic process), it is desirable to adopt [R]=K(d1), and when N=M (that realizes a stochastic process), [R]=[Formula in text] only in the first round (where * represents the population average) and [R]=K(d1) in the subsequent rounds. Based on this strategy, the mole fraction of the fittest increases by (2q)(-r) times after the rth round. With realistic parameters, we calculated several quantities such as the optimal [R] values and number of rounds needed. These values were quite reasonable and consistent with observations, suggesting the validity of our theory.

Toward an evolutionary containment of evolving pathogen-receptors by using an ensemble of multiple mutant ligands: from the viewpoint of fitness landscape in sequence space.

It is known that even if a ligand peptide is designed to bind to a target receptor on the surface of a pathogen such as viruses, bacteria or cancer cells, it is likely that some receptors are subject to random mutation and thus the ligand has a reduced ability to bind to these receptors. This issue is known as drug-resistant or escape mutants. In this paper, we present an idea to inhibit the evolving receptors by using an ensemble of all possible single- or double-point mutant sequences of the ligand peptide. Several mutant ligands in the ensemble are expected to bind to the mutant receptors, and then the ensemble may create a defensive wall surrounding the target receptors in receptor-sequence space. We examined the effectiveness of this "evolutionary containment" of the evolving receptors through eight peptide-protein complex systems, which were retrieved from the Protein Data Bank (PDB). As a result, we obtained a suggestion that the original (or parent) ligand sequence should be designed to have as high fitness as possible but to be not local optima, in order to maximize the rate of the evolutionary containment. This may be a strategy of the drug-design against evolving pathogens.

Biophysical connection between evolutionary dynamics and thermodynamics in in vitro evolution.

We analyzed a mathematical model of in vitro evolution conducted by repetition of mutagenesis and selection processes. The selection process consists of the selective enrichment and subsequent sampling as follows: each mutant with fitness W is amplified by the Boltzmann factor exp(rW/k(B)T(the)), where the fitness W is defined as the negative Gibbs free energy (-ΔG) in a reaction of the phenotypic molecules and r is the round number of the selective enrichment; then, an arbitrary mutant is randomly chosen from the resulting mutant population and it becomes a new parent in the next generation. As a result, we found that the evolutionary dynamics is described in a mathematical framework similar to thermodynamics: the "evolution constant" k(E) and "evolutionary temperature" T(evo) play key roles similar to the Boltzmann constant k(B) and thermodynamic temperature T(the), respectively. In the stationary state of the evolutionary dynamics, the attractor of the fitness is in inverse proportion to k(E)T(evo). Furthermore, beyond the mathematical analogy, we obtained a biophysical connection between evolutionary dynamics and thermodynamics. Particularly, we found that T(evo) and T(the) are connected by k(E)T(evo)≈k(B)T(the)/2r. These results suggest that we can predict the fitness value in the stationary state by the thermodynamic temperature T(the) in the experimental setup.

A mathematical consideration of the word-composition vector method in comparison of biological sequences.

To measure the similarity or dissimilarity between two given biological sequences, several papers proposed metrics based on the "word-composition vector". The essence of these metrics is as follows. First, we count the appearance frequencies of all the K-tuple words throughout each of two given sequences. Then, the two given sequences are transformed into their respective word-composition vectors. Next, the distance metrics, for example the angle between the two vectors, are calculated. A significant issue is to determine the optimal word size K. With a mathematical model of mutational events (including substitutions, insertions, deletions and duplications) that occur in sequences, we analyzed how the angle between the composition vectors depends on the mutational events. We also considered the optimal word size (=resolution) from our original approach. Our results were verified by computational experiments using artificially generated sequences, amino acid sequences of hemoglobin and nucleotide sequences of 16S ribosomal RNA.

One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

Gel shift selection of translation enhancer sequences using messenger RNA display.

We designed a new approach for selection of translation enhancer sequences that enables efficient protein synthesis in cell-free systems. The selection is based on a gel shift assay of a messenger RNA (mRNA)-protein fusion product that is synthesized in a cell-free translation system using an mRNA display method. A library of randomized 20-nt-long sequences, with all possible combinations of the four nucleotides, upstream of a coding region was screened by successive rounds of screening in which the translation time of the succeeding round was reduced compared with the previous round. An efficient translation enhancer sequence capable of more rapid initiation of cell-free protein synthesis, with a minimal translation time of 5 min, than a natural longer enhancer sequence (Xenopus ß-globin 5'UTR) was selected using rabbit reticulocyte extract as a model cell-free translation system. Furthermore, a successful screening of cap-independent translation enhancer sequence and a significant sequence similarity of the selected candidates validated the efficiency of the combined mRNA display and gel shift assay method for the rapid development of advanced cell-free translation systems.

Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer.

BACKGROUND: The microarray has contributed to developing the omic analysis. However, as it depends basically on the surface reaction, it is hard to perform bulk reactions and sequential multistep reactions. On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations. RESULTS: Here, we report a novel microarray technology which enables us to explore advanced applications, termed microarray-with-manageable volumes (MMV). The technical essence is in the pipette-free direct parallel transfer from well to well performed by centrifugation, evading the evaporation and adsorption-losses during handling. By developing the MMV plate, accompanying devices and techniques, generation of multiple conditions (256 kinds) and performance of parallel multistep reactions, including PCR and in vitro translation reactions, have been made possible. These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aß-binding or cathepsin E-inhibition. CONCLUSIONS: With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-µL scale have become possible.

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.

Biomolecular information gained through in vitro evolution.

An in vitro evolution is a simplified Darwinian evolution in well-controlled surroundings. This evolution process can be modeled as a hill-climbing or adaptive walk on a fitness landscape in sequence space. The evolving molecular system gains at least two kinds of information originating from the converged sequences and the fitness increment of the evolving biopolymer as the adaptive walker. These two represent two aspects of the biomolecular information, its extent and its content, respectively. Here, we review studies related to formulation of the "content" and "extent" of biomolecular information. The two aspects are interconnected through physicochemical properties of the biopolymer, contrary to the case of conventional information, which seems to be independent of matter. The interconnection was analyzed based on the analogy between the evolution process and thermodynamics. The linear combination of the two by a temperature-like fluctuation factor resulted in a free-energy-like monotonically increasing function during the evolution process.

cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.

We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA-protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a 'ligation site' for T4 RNA ligase, a 'biotin site' for solid-phase handling, a 'reverse transcription primer site' for the efficient and rapid conversion from 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 and a 'restriction enzyme site' for the release of a complex from the solid support. This enables not only stabilizing mRNA-protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

Development of systemic in vitro evolution and its application to generation of peptide-aptamer-based inhibitors of cathepsin E.

Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC(50). This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (P(i)101; SCGG IIII SCIA) have half an inhibition activity (K(i); 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.

Fitting protein-folding free energy landscape for a certain conformation to an NK fitness landscape.

The NK fitness landscape is a mathematical landscape model with a parameter k that governs the degree of ruggedness of the landscape. We presented a procedure to fit a given landscape to the NK fitness landscape by introducing the "apparent k-value"k(app). In this paper, we defined the protein free energy (DeltaG) landscape in amino acid sequence space, where DeltaG is the folding free energy from a random coil to a "certain conformation". Applying this landscape to our fitting procedure, we examined the statistical properties of the landscape. For calculation of a conformation energy, amino acid residues are represented by points, and interaction energies among amino acid residues are given as (1+K)-body interactions, that is, an unit of interacting (1+K) residues cooperatively contribute a single energy value to the conformational energy. Our results suggest that the apparent k-value of the free energy landscape is k(app) approximately K, and that the number of possible interactions among residues is unrelated to the k(app) value. This leads to the inference that k(app) takes values about 1-3 in real landscapes, if nature adopts two-body approximately four-body interaction energies.

Quantum dot FRET biosensors that respond to pH, to proteolytic or nucleolytic cleavage, to DNA synthesis, or to a multiplexing combination.

Fluorescent acceptors have been immobilized on nanoparticulate quantum dots (QDs), which serve in turn as their FRET donors. The broad excitation and narrow emission bands of QDs mark them as having excellent potential as donors for FRET and, in principle, differently colored QDs could be excited simultaneously. The present work describes the preparation and operation of FRET-based QD bioprobes individually able to detect the actions of protease, deoxyribonuclease, DNA polymerase, or changes in pH. In addition, two such QD-mounted biosensors were excited at a single wavelength, and shown to operate simultaneously and independently of each other in the same sample solution, allowing multiplex detection of the action of a protease, trypsin, in the presence of deoxyribonuclease.

An mRNA-protein fusion at N-terminus for evolutionary protein engineering.

A novel method to link a nascent protein (phenotype) to its mRNA (genotype) covalently through the N-terminus was developed. The mRNA harboring amber stop codon at just downstream of initiation site was hybridized with hydrazide-modified ssDNA at upstream of coding region and was ligated to the DNA. This construct was then modified with 4-acetyl-phenylalanyl amber suppressor tRNA. This modified construct was fused with the nascent protein via the phenylalanine derivative when the mRNA uses the amber suppressor tRNA to decode the amber stop codon. The obtained fusion molecule was used successfully in selective enrichment experiments. It will be applicable for high-through-put screening in evolutionary protein engineering. In contrast to fusion molecules generated by other methods in which the protein is linked to genotype molecule through the C- terminus, our fusion molecule will serve to select a protein for which the C-terminus is essential to be active.

Extracting characteristic properties of fitness landscape from in vitro molecular evolution: a case study on infectivity of fd phage to E.coli.

We have developed a methodology for extracting characteristic properties of a fitness landscape of interest by analyzing fitness data on an in vitro molecular evolution. The in vitro evolution is required to be conducted as the following "adaptive walk": a single parent sequence generates N mutant sequences as its offsprings, and the fittest individual among the N offsprings will become a new parent in the next generation. N is the library size of mutants to be screened in a single generation. Our theory of the adaptive walk on the "NK landscape" suggests the following: the adaptive walker starting from a random sequence climbs the landscape easily in an early stage, and then reaches a stationary phase in which the mutation-selection-random drift balance sets in. The stationary fitness value is nearly proportional to square root of ln N. Our analysis is performed from the following points: (1) stationary fitness values, (2) time series of fitness in the transitional state, (3) mutant's fitness distribution, and (4) the strength of selection pressure. Applying our methodology, we analyzed experimental data on the in vitro evolution of a random polypeptide (139 amino acids) toward acquiring infectivity (= ability to infect) of fd phage. As a result, we estimated that k is about 27 in this system, indicating that an arbitrary residue in a sequence is affected from other 23% residues. In this article, we demonstrated that the experimental data is consistent with our theoretical equations quantitatively, and that our methodology for extracting characteristic properties of a fitness landscape may be effective.

Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library.

A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.