Lipid vesicle-based molecular robots.

A molecular robot, which is a system comprised of one or more molecular machines and computers, can execute sophisticated tasks in many fields that span from nanomedicine to green nanotechnology. The core parts of molecular robots are fairly consistent from system to system and always include (i) a body to encapsulate molecular machines, (ii) sensors to capture signals, (iii) computers to make decisions, and (iv) actuators to perform tasks. This review aims to provide an overview of approaches and considerations to develop molecular robots. We first introduce the basic technologies required for constructing the core parts of molecular robots, describe the recent progress towards achieving higher functionality, and subsequently discuss the current challenges and outlook. We also highlight the applications of molecular robots in sensing biomarkers, signal communications with living cells, and conversion of energy. Although molecular robots are still in their infancy, they will unquestionably initiate massive change in biomedical and environmental technology in the not too distant future.

Simultaneous Recognition of Over- and Under-Expressed MicroRNAs Using Nanopore Decoding.

This paper describes a strategy for simultaneous recognition of over- and under-expressed microRNAs (miRNAs) using the method of signal classification-based nanopore decoding. MiRNA has attracted attention as a promising biomarker for cancer diagnosis owing to its cancer-type-specific expression patterns. While nanopore technology has emerged as a simple and label-free method to detect miRNAs and their expression patterns, recognizing patterns involving simultaneous over/under-expression is still challenging due to the inherent working principles. Here, inspired by the sequence design for DNA computation with nanopore decoding, we designed diagnostic DNA probes targeting two individual over/under-expressed miRNAs in the serum of oral squamous cell carcinoma. Through nanopore measurements, our designed probes exhibited characteristic current signals depending on the hybridized miRNA species, which were plotted on the scatter plot of duration versus current blocking ratio. The classified signals reflected the relative abundance of target miRNAs, thereby enabling successful pattern recognition of over/under-expressed miRNAs, even when using clinical samples. We believe that our method paves the way for miRNA-targeting simple diagnosis as a liquid biopsy.

MicroRNA Detection at Femtomolar Concentrations with Isothermal Amplification and a Biological Nanopore.

Nanopore sensing is a powerful tool for the rapid and label-free detection of oligonucleotides, including microRNA. When moving towards actual diagnostic applications, detection of microRNA at low concentrations is one of the significant issues to be addressed. We here describe a method to detect ultra-low concentrations of microRNA using isothermal amplification and nanopore technology. Using this method, the amplified DNA from 1 fM of target microRNA can be measured by a nanopore measurement.

Single polypeptide detection using a translocon EXP2 nanopore.

DNA sequencing using nanopores has already been achieved and commercialized; the next step in advancing nanopore technology is towards protein sequencing. Although trials have been reported for discriminating the 20 amino acids using biological nanopores and short peptide carriers, it remains challenging. The size compatibility between nanopores and peptides is one of the issues to be addressed. Therefore, exploring biological nanopores that are suitable for peptide sensing is key in achieving amino acid sequence determination. Here, we focus on EXP2, the transmembrane protein of a translocon from malaria parasites, and describe its pore-forming properties in the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled from 7 monomers. Using the EXP2 nanopore allowed us to detect poly-L-lysine (PLL) at a single-molecule level. Furthermore, the EXP2 nanopore has sufficient resolution to distinguish the difference in molecular weight between two individual PLL, long PLL (Mw: 30,000-70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide-detectable nanopores.

Nanopore decoding for a Hamiltonian path problem.

DNA computing has attracted attention as a tool for solving mathematical problems due to the potential for massive parallelism with low energy consumption. However, decoding the output information to a human-recognizable signal is generally time-consuming owing to the requirement for multiple steps of biological operations. Here, we describe simple and rapid decoding of the DNA-computed output for a directed Hamiltonian path problem (HPP) using nanopore technology. In this approach, the output DNA duplex undergoes unzipping whilst passing through an α-hemolysin nanopore, with information electrically decoded as the unzipping time of the hybridized strands. As a proof of concept, we demonstrate nanopore decoding of the HPP of a small graph encoded in DNA. Our results show the feasibility of nanopore measurement as a rapid and label-free decoding method for mathematical DNA computation using parallel self-assembly.