Nanopore based single-molecule temporal omics

The various functions and activities of the living system involve the coordination of a series of complicated molecular systems, based on which, omics aiming at characterizing and quantifying a large library of biomolecules has been attracting increasing attentions. At present, omics is mainly based on massively parallel sequencing and mass spectrometry (MS) combined with chromatography (GC, LC, etc.). However, as life research enters the single-cell level and the scope of research continues to expand, the heterogeneity of single cells, the low sample quantity of single cell for omics analysis, the low-abundance metabolites that play a key role in disease characteristics, and the rare mutations bring new challenges to the current omics approaches. Single-molecule techniques have greatly increased our understanding of the complex molecular regulatory networks and mechanisms in biological systems. Traditional single-molecule approaches, such as single-molecule force spectrometry (AFM, tweezers, etc.), commonly catch a single molecule for a long time, therefore lacking the capability for identifying a large number of biomolecules in high-throughput. The requirements for labelling also limited the wide applications of fluorescence-based approach for omics analysis. Moreover, due to the presence of parallel and cascading reactions, enzymatic reactions exhibit temporal dynamics, and intermediate molecules with low abundance and short half-life are often lost in breakpoint sampling and average measurements of large systems. Therefore, it is urgent to develop a single-molecule approach with high throughput for the single-molecule and temporal dynamic omics to obtain the heterogeneity and dynamics of biological processes. In this view, we explore the potential advantages of nanopore single-molecule approach for omics research. Based on the recording the change in ionic current through the nanopore with the target molecule inside, nanopore could characterize individual molecules one by one. Therefore, nanopore approach owns the intrinsic sensing capability with high spatial resolution, single molecule and high throughput. Accordingly, the corresponding advances of nanopores from these three perspectives were highlighted and discussed with three aspects: (1) "Single-molecule mass spectrometry" for directly reading the single unit differences within biopolymers from single-molecule perspective, including DNA and peptide, allowing the identifying the biomolecule in low abundance; (2) revealing single-molecule heterogeneity for uncovering the heterogeneous conformation and modifications of individual molecules, including the "open" and "stack" conformers of flavin adenine dinucleotide (FAD) and the different phosphorylation site in Tau peptide; (3) real-time monitoring the bioprocesses for unveiling the temporal dynamics of enzymatic reactions, such as obtaining the stepwise cleavage of ssDNA by exonuclease I and studying the crosstalk effects between enzymes. Furthermore, we propose the concept of "single-molecule temporal omics" based on nanopore, which achieves omics analysis at the single-molecule level with low time intervals. Combined with existing "spatial omics," nanopore based single-molecule temporal omics would help to provide a spatiotemporal evolution spectrum of biological molecules with high coverage and depth to address new biological problems.