Site-Specific Introduction of Bioorthogonal Handles to Nanopores by Genetic Code Expansion

Site-specific functionalization of natural amino acid-containing biological nanopores is pivotal in single molecule sensing. However, pore engineering methodologies are restricted to a limited choice and introduction of unnatural chemical components is extremely difficult. Herein we report the genetic code expansion (GCE) strategy to introduce unnatural amino acid (UAA) to an octameric Mycobacterium smegmatis porin A (MspA) nanopore. GCE allows for rapid and efficient introduction of bioorthogonal reactive site (i.e., azide) to the pore rim, and conjugation of single stranded DNA or lysozyme was demonstrated. The lysozyme-conjugated pore was further used for the discrimination of different oligosaccharides, demonstrating a sensing capacity that a bare MspA nanopore does not possess. GCE with bioorthogonal handles, which has never been previously applied in the preparation of nanopores, is a versatile strategy for pore engineering and may further expand the application scenarios of nanopores.     

Third-Generation Sequencing of Epigenetic DNA

The discovery of epigenetic bases has revolutionised the understanding of disease and development. Among the most studied epigenetic marks are cytosines covalently modified at the 5 position. In order to gain insight into their biological significance, the ability to determine their spatiotemporal distribution within the genome is essential. Techniques for sequencing on “next-generation” platforms often involve harsh chemical treatments leading to sample degradation. Third-generation sequencing promises to further revolutionise the field by providing long reads, enabling coverage of highly repetitive regions of the genome or structural variants considered unmappable by next generation sequencing technology. While the ability of third-generation platforms to directly detect epigenetic modifications is continuously improving, at present chemical or enzymatic derivatisation presents the most convenient means of enhancing reliability. This Review presents techniques available for the detection of cytosine modifications on third-generation platforms.     

A Duplex Polymerization Strategy for General,Programmable and High-Resolution Nanopore-Based Sensing

Nanopore sensing is highly promising in single molecular analysis but their broad applications have been challenged by the limited strategies that can transduce a target-of-interest into a specific and anti-false/inference signal, especially for solid-state nanopores with relatively lower resolution and higher noise. Here we report a high-resolution signal-production concept named target-induced duplex polymerization strategy (DPS). Through linking the same or different duplex substrates (DSs) with a special linker (L) and an optional structure tag (ST), the DPS can generate target-specific DS polymers with highly controllable duration times, duration intervals and even distinguished secondary tagging currents. Experimentally, DPS mono-polymerization of single DS and co-polymerization of multiple DSs has verified the duration time of a DPS product is the sum of those for each DS monomer. Tetrahedron-DNA structures with different sizes are used as the STs to provide needle-like secondary peaks for further resolution enhancement and multiplex assay. With these examples DPS represents a general, programmable and advanced strategy that may simultaneously provide size-amplification, concentration amplification, and signal-specificity for molecular recognition. It is also promisingly in various applications regarding to single molecular investigation, such as polymerization degree, structure/side chain conformation, programmable multiplex decoding and information index.