Yuzuru Husimi’s research while affiliated with The Graduate University for Advanced Studies, SOKENDAI and other places

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.

The norovirus RNA replicase (NV3Dpol, 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 Qβ 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 NV3Dpol, 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 17th round of transfer gave a main peak around ---CAAC-3′. In the preceding studies on the batch amplification reaction starting from a singlestranded 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 NV3Dpol.

Biological evolution at the molecular level is conceptually regarded as the genetic information gaining process. Analyzing the in vitro evolution process, which is a simplified Darwinian evolution under a well-controlled environment, we can clarify the concept of the information gaining process. This evolution process can be modeled as a hill-climbing or adaptive walk on a fitness landscape in sequence space. Through the hill-climbing process, the evolving biopolymer (as the adaptive walker) stores the following two aspects of information: one stems from the sequences converged in sequence space and the other stems from the fitness increment on the fitness landscape. In Eigen’s words, the former and latter are described as the “extent” and “content” of biological information, respectively [25]. In our approach, these two aspects can be interpreted based on the analogy between evolutionary dynamics and thermodynamics. Several studies introduced the concept of “free fitness” (which is analogous to free energy) as the Lyapunov function for evolution: Free fitness ≡ Fitness + Temperature − like parameter ×Entropy. Furthermore, we focus on the novel quantity of Fitness divided by \mbox{\it Temperature-like para\-meter}, and regard this quantity as the content of information, while we regard Entropy as the extent of information. The quantity of Free fitness divided by Temperature-like parameter is a Lyapunov function of the evolution process, and then it should be called “biomolecular information”, which includes both aspects of information.

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.

The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Qbeta replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3Dpol) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3Dpol in vitro were re-evaluated in this context. NV3Dpol, 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, NV3Dpol 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. NV3Dpol 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 NV3Dpol in vitro. NV3Dpol can be used as an RNA replicase in in vitro RNA + protein evolution with the RNA of special terminal sequences.