Abstract: Methods of characterizing an analyte using a nanopore. One aspect features methods for characterizing a double-stranded polynucleotide using a nanopore, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.

Abstract: Provided herein relate to modified or mutant forms of cytolysin A (ClyA) and compositions comprising the same. In particular, the modified or mutant forms of ClyA permits efficient capture and/or translocation of a target negative-charged molecule or polymer through the modified or mutant ClyA nanopores at low or physiological ionic strengths. Thus, methods for using the modified or mutant forms of ClyA and compositions, for example, for characterizing a target negatively-charged analyte, e.g., a target polynucleotide, are also provided.