![]() ![]() ![]() As the RNA molecule passes through a constriction point within the central channel of the nanopore (the “reader head”), changes in the flow of ions create local alterations in electric current signals that are sensed by an embedded ammeter. In direct RNA sequencing, the 3′-ends of single-stranded native RNA or the RNA strand of a cDNA/RNA hybrid are ligated to a sequencing adapter that has been pre-loaded with a helicase (A) When introduced into the nanopore flow cell, the helicase docks with one of thousands of individual nanopores embedded within a charged membrane, and threads its nucleic acid cargo into the central channel of the nanopore at an average speed of approximately 70 bases per second ( Garalde et al., 2018). However, these motor protein activities are stochastic, meaning that the time intervals between each stepwise advance of the DNA or RNA molecule are variable ( Deamer et al., 2016), with current helicases averaging ∼70 bases per second for RNA ( Garalde et al., 2018) and up to 450 bases per second for DNA when coupled with an R9.4 nanopore ( Wang Y. ![]() While early proof of principle experiments used DNA polymerases to slow down this translocation process ( Cherf et al., 2012 Manrao et al., 2012), the current commercial solution from Oxford Nanopore Technologies (ONT) employs an engineered helicase enzyme to both unwind double stranded molecules and introduce single stranded nucleic acid into the nanopore sensor at a controlled rate for sequencing ( Figure 1A). By embedding the nanopore within a membrane with a constant voltage bias, an ionic current drives single stranded nucleic acids through the pore ( Clamer et al., 2014) at the narrowest aperture of this pore (the “reader head”), the flow of ions is differentially suppressed depending on the size and shape of the nucleobases present ( Cherf et al., 2012 Manrao et al., 2012 Smith et al., 2015). As this technology matures, we anticipate advances in both sequencing chemistry and analysis methods will lead to rapid improvements in the identification and quantification of these epigenetic marks.įirst conceptualized in the 1980s ( Tobkes et al., 1985), nanopore sequencing uses a modified transmembrane protein (the nanopore) as both a channel through which a nucleic acid passes, and a biosensor capable of sensing the nucleobase content of that nucleic acid ( Deamer et al., 2016). This review is intended to introduce the reader to nanopore sequencing and key principles underlying its use in direct detection of nucleic acid modifications in unamplified DNA or RNA samples, and outline current approaches for detecting and quantifying nucleic acid modifications by nanopore sequencing. To date, more than a dozen endogenous DNA and RNA modifications have been interrogated by nanopore sequencing, as well as a number of synthetic nucleic acid modifications used in metabolic labeling, structure probing, and other emerging applications. While the former can indirectly sense modified nucleotides through changes in the kinetics of reverse transcription reactions, nanopore sequencing can in principle directly detect any nucleic acid modification that produces a signal distortion as the nucleic acid passes through a nanopore sensor embedded within a charged membrane. Third generation sequencing technologies such as the commercially available “long read” platforms from Pacific Biosciences and Oxford Nanopore Technologies are an attractive alternative for high throughput detection of nucleic acid modifications. Moreover, such approaches tend to be specific to a single class of RNA or DNA modification, and generate only indirect readouts of modification status. However, the majority of nucleic acid modifications lack commercial monoclonal antibodies, and mapping techniques that rely on chemical or enzymatic treatments to manipulate modification signatures add additional technical complexities to library preparation. For nucleic acid modifications, NGS has been coupled with immunoprecipitation, chemical treatment, enzymatic treatment, and/or the use of reverse transcriptase enzymes with fortuitous activities to enrich for and to identify covalent modifications of RNA and DNA. Next generation sequencing (NGS) has provided biologists with an unprecedented view into biological processes and their regulation over the past 2 decades, fueling a wave of development of high throughput methods based on short read DNA and RNA sequencing. Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora, CO, United States. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |