RESUMEN
A single nucleotide variant present between two otherwise identical nucleic acids will have unexpected functional consequences frequently. Here, a neoteric single nucleotide variation (SNV) detection assay that integrates two complementary nanotechnology systems, nanoassembly technology and an ingenious nanopore biosensing platform, has been applied to this research. Specifically, we set up a detection system to reflect the binding efficiency of the polymerase and nanoprobe through the difference of nanopore signals and then explore the effect of base mutation at the binding site. In addition, machine learning based on support vector machines is used to automatically classify characteristic events mapped by nanopore signals. Our system reliably discriminates single nucleotide variants at binding sites, even possessing the recognition among transitions, transversions, and hypoxanthine (base I). Our results demonstrate the potential of solid-state nanopore detection for SNV and provide some ideas for expanding solid-state nanopore detection platforms.
Asunto(s)
Nanoporos , Ácidos Nucleicos , Nucleótidos , ADN/química , ADN Polimerasa Dirigida por ADN , Nanotecnología/métodosRESUMEN
As biological macromolecules, proteins are involved in important cellular functions ranging from DNA replication and biosynthesis to metabolic signalling and environmental sensing. Protein sequencing can help understand the relationship between protein function and structure, and provide key information for disease diagnosis and new drug design. Nanopore sensors are a novel technology to achieve the goal of label-free and high-throughput protein sequencing. In recent years, nanopore-based biosensors have been widely used in the detection and analysis of biomolecules such as DNA, RNA, and proteins. At the same time, computer simulations can describe the transport of proteins through nanopores at the atomic level. This paper reviews the applications of nanopore sensors in protein sequencing over the past decade and the solutions to key problems from a computer simulation perspective, with the aim of pointing the way to the future of nanopore protein sequencing.
Asunto(s)
Técnicas Biosensibles , Nanoporos , Simulación por Computador , Proteínas , ADN/química , NanotecnologíaRESUMEN
Proteins have a small volume difference by the diversity of amino acids, which make protein detection and identification a great challenge. Solid-state nanopore as label-free biosensors has attracted attention with high sensitivity. In this work, we investigated the Taq DNA polymerase before and after combining it with a DNA substrate on a solid-state nanopore through molecular dynamics. In simulation, we analyzed the contribution source of nanopore current blockage. In addition to considering the traditional physical exclusion volume model, the non-covalent interaction between the protein molecules and the pore wall also showed to affect the current blockage in the nanopore. When choosing pores of comparable size to protein molecules, the two states of Taq DNA polymerase produce differentiated non-covalent interactions with the pore wall, which enhanced the amplitude difference in current blockage. As a result, the two DNA polymerases can be distinguished through the distinct current blockage. However, when applying additional pulling force or increasing the pore size of the nanopore, the differences between the current blockages are not significant enough to distinguish. The introduction of the non-covalent interaction makes it clear to understand the current blockage differences, which guide the mechanism between molecules with similar structures or volumes.
Asunto(s)
Técnicas Biosensibles , Nanoporos , Simulación de Dinámica Molecular , Polimerasa Taq/metabolismo , ADN/químicaRESUMEN
The investigation of abnormal experimental phenomena observed in nanopore research improves our understanding of nanopores. In this article, we report and explore the unusual phenomenon that the amplitude of current blockage decreases beyond zero baseline (overflow amplitudes), which was observed in the translocation behavior of 100 bp double-stranded DNA molecules through SiNx nanopores. In our experiments, the overflow amplitude decreases with the increase of salt concentration and also decreases when the dwell time is shortened as the normalized amplitude of the overflow current showed a reduction with the increase of voltage. Upon analyzing the electric double layer meanwhile, the overflow amplitudes were shown to be positively correlated with the depth of the electric double layer and the duration of interaction between biological molecules. The formation of overflow amplitude can be attributed to the double electric layer ionic perturbation and reconfiguration, which are the results of the interaction between the biomolecule and the electric bilayer. The validation of the assumption using biomolecules containing different charges demonstrated that the overflow amplitude increased with the increase of the charge. It was concluded that proteins that pass through the nanopore with different orientation were differentiated based on their different overflow amplitude patterns. The investigation of overflow amplitude helps to enhance the understanding and the performance of nanopores.
Asunto(s)
Nanoporos , ADN/metabolismo , Iones , ElectricidadRESUMEN
We developed EasyNanopore which is a ready-to-use software to select the events of a nanopore molecular translocation experiment. The software is released as an executable file with a graphical user interface and provides several versions suitable for different operating systems without installing any running environment to execute it. We use the adaptive threshold which adapts to the low-frequency variation of the baseline to detect events and uses a multiprocess method to accelerate the process of event detection. After the event is identified, its duration and amplitude information will be extracted and a resulting txt file will be generated for further analysis. Our software runs fast and can effectively extract the data from data of large-scale nanopore molecular translocation experiments.