Device and method for analyzing target

Patent metadata

Field	Value
Publication number	US-9880174-B2
Application number	US-201214387389-A
Country	US
Kind code	B2
Filing date	Dec 12, 2012
Priority date	Mar 23, 2012
Publication date	Jan 30, 2018
Grant date	Jan 30, 2018

Abstract

The present invention provides a novel sensor for detecting a target. The nucleic acid sensor of the present invention includes a nucleic acid element that includes a catalyst nucleic acid molecule (D) that exerts a catalytic function and a binding nucleic acid molecule (A) that binds to a target. The nucleic acid element is a double-stranded nucleic acid element including a first strand and a second strand. The first strand (ss1) includes the binding nucleic acid molecule (A), a loop-forming sequence (L1), and the catalyst nucleic acid molecule (D) linked in this order. The second strand (ss2) includes a stem-forming sequence (S A ), a loop-forming sequence (L2), and a stem-forming sequence (S D ) linked in this order. In this nucleic acid element, in the absence of a target, the catalytic function of the catalyst nucleic acid molecule (D) is inhibited by stem formation in each of the stem-forming sequences (S A ) and (S D ), and in the presence of ATP/target, the stem formation is released by a binding of the binding nucleic acid molecule (A) with the target, and the catalytic function is exerted.

First claim

The invention claimed is:

1. A nucleic acid sensor comprising at least one nucleic acid element chosen from (I) and (II) that comprises a catalyst nucleic acid molecule (D) and a binding nucleic acid molecule (A),
(I) a single-stranded nucleic acid element comprising the binding nucleic acid molecule (A), a loop-forming sequence (L1), a stem-forming sequence (S D ), the catalyst nucleic acid molecule (D), a loop-forming sequence (L2), and a stem-forming sequence (S A ) linked in this order,
wherein
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (S A ),
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (S D ),
the loop-forming sequence (L1) is non-complementary to the loop-forming sequence (L2), and
wherein,
in the absence of a target, the catalyst nucleic acid molecule (D) is caged by stem formation in each of the stem-forming sequences (S A ) and (S D ),
in the presence of a target, the stem-forming sequence (S A ) and the stem-forming sequence (S D ) are released from the binding nucleic acid molecule (A) and the catalyst nucleic acid molecule (D), and the catalyst nucleic acid molecule (D) is released,
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (S A ) form a stem,
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side and the stem-forming sequence (S D ) form a stem, and
the loop-forming sequences (L1) and (L2) form an internal loop between the two stems;

(II) a single-stranded nucleic acid element comprising the catalyst nucleic acid molecule (D), a loop-forming sequence (L2), a stem-forming sequence (S A ), the binding nucleic acid molecule (A), a loop-forming sequence (L1), and a stem-forming sequence (S D ) linked in this order,
wherein
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side is complementary to the stem-forming sequence (S D ),
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side is complementary to the stem-forming sequence (S A ),
the loop-forming sequence (L1) is non-complementary to the loop-forming sequence (L2), and
wherein,
in the absence of a target, the catalyst nucleic acid molecule (D) is caged by stem formation in each of the stem-forming sequences (S A ) and (S D ),
in the presence of a target, the stem-forming sequence (S A ) and the stem-forming sequence (S D ) are released from the binding nucleic acid molecule (A) and the catalyst nucleic acid molecule (D), and the catalyst nucleic acid molecule (D) is released,
a terminal region of the binding nucleic acid molecule (A) on the loop-forming sequence (L1) side and the stem-forming sequence (S A ) form a stem,
a terminal region of the catalyst nucleic acid molecule (D) on the loop-forming sequence (L2) side and the stem-forming sequence (S D ) form a stem, and
the loop-forming sequences (L1) and (L2) form an internal loop between the two stems,

further wherein the catalyst nucleic acid molecule (D) exerts an oxidation-reduction reaction and the target is ATP or AMP.

2. The nucleic acid sensor according to claim 1 , wherein
the nucleic acid element (I) comprises, from the 3′ side thereof, the binding nucleic acid molecule (A), the loop-forming sequence (L1), the stem-forming sequence (S D ), the catalyst nucleic acid molecule (D), the loop-forming sequence (L2), and the stem-forming sequence (S A ) linked in this order,
a 5′ terminal region of the binding nucleic acid molecule (A) is complementary to the stem-forming sequence (S A ), and
a 5 terminal region of the catalyst nucleic acid molecule (D) is complementary to the stem-forming sequence (S D ).

3. The nucleic acid sensor according to claim 2 , wherein
the length of each of the loop-forming sequences (L1) and (L2) ranges from 1- to 30-nucleotides.

4. The nucleic acid sensor according to claim 2 , wherein
the length of the stem-forming sequence (S A ) ranges from 1- to 60-nucleotides, and
the length of the stem-forming sequence (S D ) ranges from 1- to 30-nucleotides.

5. The nucleic acid sensor according to claim 1 , wherein
the length of the binding nucleic acid molecule (A) ranges from 18- to 60-nucleotides.

6. The nucleic acid sensor according to claim 1 , wherein
the binding nucleic acid molecule (A) comprises the following polynucleotide (a1), (a2), (a3), or (a4):
(a1) a polynucleotide composed of a base sequence of SEQ ID NO: 1,
(a2) a polynucleotide that is composed of a base sequence obtained by substitution, deletion, addition and/or insertion of at least one base in the base sequence of the polynucleotide (a1),
(a3) a polynucleotide that is composed of a base sequence with 50% or more identity with the base sequence of the polynucleotide (a1), and
(a4) a polynucleotide that is composed of a base sequence complementary to a base sequence that hybridizes to the base sequence of the polynucleotide (a1) under stringent conditions.

7. The nucleic acid sensor according to claim 1 , wherein
the length of the catalyst nucleic acid molecule (D) ranges from 15- to 30-nucleotides.

8. The nucleic acid sensor according to claim 1 , wherein
the catalyst nucleic acid molecule (D) comprises the following polynucleotide (d1), (d2), (d3), or (d4):
(d1) a polynucleotide composed of a base sequence of any of SEQ ID NOs: 11 to 31 and 66 to 85,
(d2) a polynucleotide that is composed of a base sequence obtained by substitution, deletion, addition, and/or insertion of at least one base in the base sequence of the polynucleotide (d1) that exerts the oxidation-reduction reaction,
(d3) a polynucleotide that is composed of a base sequence with 50% or more identity with the base sequence of the polynucleotide (d1) that exerts the oxidation-reduction reaction, and
(d4) a polynucleotide that is composed of a base sequence complementary to a base sequence that hybridizes to the base sequence of the polynucleotide (d1) under stringent conditions that exerts the oxidation-reduction reaction.

9. A device for analyzing a target, comprising:
a base material;
a nucleic acid sensor; and
a detection part, wherein
the nucleic acid sensor and the detection part are disposed on the base material,
the nucleic acid sensor is the nucleic acid sensor according to claim 1 , and
the detection part is a detection part that detects when the catalyst nucleic acid molecule (D) in the nucleic acid sensor is released.

10. The device according to claim 9 , wherein the nucleic acid sensor is linked with the base material via a linker.

11. The device according to claim 9 , wherein the nucleic acid sensor is disposed on the detection part.

12. The device according to claim 9 , wherein the detection part detects a signal generated when the catalyst nucleic acid molecule (D) is released.

13. The device according to claim 12 , wherein the signal is an optical signal or an electrochemical signal.

14. The device according to claim 9 , further comprising:
a reagent part, wherein the reagent part comprises a substrate that reacts with the catalyst nucleic acid molecule (D).

15. A method for analyzing a target, comprising:
a contact step of causing a sample to be in contact with the nucleic acid sensor according to claim 1 ; and
a detection step of detecting that the catalyst nucleic acid molecule (D) in the nucleic acid sensor is released.

Assignees

• Horii Katsunori
• Kaneko Naoto
• Akitomi Jou
• Katou Shintarou
• Waga Iwao
• Nec Solution Innovators Ltd

Inventors

• Horii Katsunori
• Kaneko Naoto
• Akitomi Jou
• Katou Shintarou
• Waga Iwao

Classifications

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C12Q1/008 Primary

  for determining co-enzymes or co-factors, e.g. NAD, ATP · CPC title

• G01N33/6812

  Assays for specific amino acids · CPC title

• G01N21/59

  Transmissivity (G01N21/25 takes precedence) · CPC title

• C12Y111/01007

  Peroxidase (1.11.1.7), i.e. horseradish-peroxidase · CPC title