Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.
Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing – information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies’ sequencing technology is capable of electronically analysing a sample’s DNA directly, and in real-time. In this manuscript we demonstrate the ability of an array of nanopores to sequence RNA directly, and we apply it to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analysing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.
Cooperative reactivity in solution is common in biological systems but not in purely chemical systems.1•2 In its simplest form, cooperativity is displayed when multiple identical chemical sites communicate by an allosteric mechanism so that reaction at one of the sites makes the same reaction at a second site more favorable.3 45This process can be accomplished either by ligandinduced aggregation or by ligand-induced conformational change in a preformed aggregate or other multisite system. The few attempts to model allosteric effects with small molecules have dealt with the latter mechanism.4-6 We now describe a class of model metalloporphyrin compounds that display cooperativity through ligand-induced dimerization.The most thoroughly studied cooperative system is hemoglobin, in which four almost identical heme sites react with either dioxygen or carbon monoxide as described above.7 Hemoglobin has been
Chemistry is in a powerful position to synthetically replicate biomolecular structures. Adding functional complexity is key to increase the biomimetics’ value for science and technology yet is difficult to achieve with poorly controlled building materials. Here, we use defined DNA blocks to rationally design a triggerable synthetic nanopore that integrates multiple functions of biological membrane proteins. Soluble triggers bind via molecular recognition to the nanopore components changing their structure and membrane position, which controls the assembly into a defined channel for efficient transmembrane cargo transport. Using ensemble, single-molecule, and simulation analysis, our activatable pore provides insight into the kinetics and structural dynamics of DNA assembly at the membrane interface. The triggered channel advances functional DNA nanotechnology and synthetic biology and will guide the design of controlled nanodevices for sensing, cell biological research, and drug delivery.
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