Adapter molecule, biomolecule-adapter molecule complex in which said adapter molecule and biomolecule are bound, biomolecule analysis apparatus, and biomolecule analysis method
A double-stranded DNA to be analyzed is analyzed without subjecting it to a modification treatment. An adapter molecule to be bound to the double-stranded DNA to be analyzed has a double-stranded nucleic acid region having base sequences complementary to each other, a pair of single-stranded nucleic acid regions having base sequences non-complementary to each other, and a block molecule placed in one of the single-stranded nucleic acid regions.
1 . A method of analyzing a biomolecule, comprising:
a step of applying a voltage to between a first liquid tank and a second liquid tank, which are placed opposite to each other via a thin film having a nanopore, while filling the first liquid tank with an electrolyte solution containing a bio-adapter molecule complex and filling the second liquid tank with an electrolyte solution to form a potential gradient between the first liquid tank and the second liquid tank with the first liquid tank as a negative or ground potential and the second liquid tank as a positive potential; and
a step of measuring a signal generated when the bio-adapter molecule complex passes through the nanopore from the second liquid tank to the first liquid tank;
wherein in the step of forming a potential gradient, a single-stranded nucleic acid region of the bio-adapter molecule complex is introduced into the second liquid tank via the nanopore and the bio-adapter molecule complex transfers from the first liquid tank to the second liquid tank by the potential gradient formed between the first liquid tank and the second liquid tank,
wherein the bio-adapter molecule complex has a molecular motor binding site in a single-stranded nucleic acid region having a 3′ end as a terminal portion thereof and has a primer binding site on the side of the 3′ end relative to the molecular motor binding site,
the electrolyte solution with which the first liquid tank is filled contains a molecular motor which can bind to the molecular motor binding site,
the bio-adapter molecule complex has a spacer to which the molecular motor cannot be bound between the molecular motor binding site and the primer binding site,
the bio-adapter molecule complex has a first block molecule having a diameter larger than the diameter of the nanopore placed in a single-stranded nucleic acid region having a 3′ end as a terminal portion thereof, a second block molecule having a diameter smaller than the diameter of the nanopore binds to a 5′ end of the single-stranded nucleic acid region,
the single-stranded nucleic acid region having the 5′ end is introduced into the second liquid tank, and the first block molecule placed in the single-stranded nucleic acid region having the 3′ end having the molecular motor binding site is brought into contact with the nanopore to stop the transfer of the bio-adapter molecule complex from the first liquid tank to the second liquid tank.
2 . The method of analyzing a biomolecule according to claim 1 , wherein the single-stranded nucleic acid region having the 5′ end as a terminal portion thereof is introduced into the second liquid tank via the nanopore.
3 . The method of analyzing a biomolecule according to claim 1 , wherein
the molecular motor binds to the molecular motor binding site and the molecular motor transfers the bio-adapter molecule complex from the second liquid tank to the first liquid tank.
4 . The method of analyzing a biomolecule according to claim 1 , wherein
the electrolyte solution with which the first liquid tank is filled contains a primer which can be hybridized with the primer binding site, and
the molecular motor synthesizes a complementary strand from the primer hybridized with the primer binding site and thereby transfers the bio-adapter molecule complex from the second liquid tank to the first liquid tank.
5 . The method of analyzing a biomolecule according to claim 1 , wherein
the molecular motor in contact with the nanopore synthesizes a complementary strand from the primer hybridized with the primer binding site and thereby transfers the bio-adapter molecule complex from the second liquid tank to the first liquid tank.
6 . The method of analyzing a biomolecule according to claim 1 , wherein in the step of measuring a signal, a voltage applied to between the first liquid tank and the second liquid tank is reversed when a signal from a specific region of the adapter molecule is measured and a potential gradient is formed with the first liquid tank as a positive potential and with the second liquid tank as a negative or ground potential.
7 . The method of analyzing a biomolecule according to claim 1 , wherein
the second block molecule that binds to the 5′ end of the single-stranded nucleic acid region is introduced into the second liquid tank via the nanopore.
8 . The method of analyzing a biomolecule according to claim 1 , wherein the step of measuring a signal is repeated by controlling a voltage to be applied to between the first liquid tank and the second liquid tank and thereby reciprocating the bio-adapter molecule complex between the first liquid tank and the second liquid tank.
9 . The method of analyzing a biomolecule according to claim 1 , wherein the first block molecule and the second block molecule are selected from avidin, streptavidin, or a complex between an anti-DIG antibody and a bead.