Compositions, systems, and methods for sequencing polynucleotides using tethers anchored to polymerases adjacent to nanopores

A composition including:

• a nanopore including a first side, a second side, and an aperture extending through the first and second sides;
• a plurality of nucleotides, wherein each of the nucleotides comprises an elongated tag;
• first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide;
• a polymerase disposed adjacent to the first side of the nanopore, the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide;
• a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, the head region being anchored to the polymerase, wherein the elongated body is disposed in the aperture of the nanopore;
• a first moiety disposed on the elongated body, wherein the first moiety is configured to bind to the elongated tag of a first nucleotide upon which the polymerase is acting;
• and
• a reporter region disposed on the elongated body, wherein the reporter region is configured to indicate when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide.