# US 11,993,806 B2 · Method of detecting or method of quantifying oligonucleotides

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- Number: US 11,993,806 B2
- Document ID: US11993806B2
- Status: Granted
- Kind: B2
- Application number: 17/274,813
- Filed: 2019-09-10
- Published: 2022-02-17
- Granted: 2024-05-28
- Earliest claimed: 2018-09-11
- Assignees: Sekisui Medical Co., Ltd.
- Applicants: Sekisui Medical Co., Ltd.
- Inventors: Akane Oomori; Koichi Shibusawa
- Web page: https://patentgrep.com/patent/US11993806B2

## CPC

- C12Q 1/682: Signal amplification
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention provides a method of detecting and a method of quantifying oligonucleotides with more excellent specificity and quantitativity as compared to conventional signal amplification (PALSAR) measurement methods. In the present invention, the problem is solved by hybridizing a target oligonucleotide to be measured with a complementary nucleic acid probe (3′-complementary sequence of target sequence-5′), or hybridizing the target oligonucleotide to be measured having given bases such as poly(A) added thereto with a complementary nucleic acid probe (3′-complementary sequence of target oligonucleotide+complementary sequence of given bases-5′), decomposing and removing an incomplete hybridization product by using a single-strand-specific nuclease such as Si nuclease, and measuring the nucleic acid probe contained in a remaining complete hybridization product by a PALSAR method.

## Claims

### Claim 1

A method of detecting a target oligonucleotide in a sample, comprising steps of:
(i) bringing a capture probe for capturing the target oligonucleotide into contact with the sample,
the capture probe containing
(A) a nucleic acid probe, and
(B) a solid phase, or an adapter or a linker, adjacent to a nucleotide at the 3′ end or 5′ end of the nucleic acid probe,
the nucleic acid probe containing a sequence complementary to a complete or partial sequence of the target oligonucleotide;
(ii) bringing a single-strand-specific nuclease into contact with the sample subjected to the contact step with the capture probe;
(iii) making the capture probe in the sample single-stranded after the contact step with the single-strand-specific nuclease is performed;
(iv) bringing a pair of probes capable of self-aggregation composed of first and second oligonucleotides into contact with the sample subjected to the step of making the capture probe single-stranded to form a complex of the capture probe and an oligonucleotide polymer generated by self-aggregation of the first and second oligonucleotides; and
(v) detecting the complex, and thereby detecting the target oligonucleotide in the sample.

### Claim 2 (depends on 1)

The method of detecting a target oligonucleotide according to claim 1, wherein at least one oligonucleotide of the pair of probes capable of self-aggregation composed of the first and second oligonucleotides contains the complete or partial sequence of the target oligonucleotide.

### Claim 3 (depends on 1)

The method of detecting a target oligonucleotide according to claim 1, wherein
the complex forming step of (iv) includes
a step of bringing into contact with an assist probe containing both the complete or partial sequence of the target oligonucleotide and a sequence complementary to a complete or partial sequence of at least one of the first and second oligonucleotides.

### Claim 4

A method of detecting a target oligonucleotide in a sample, comprising steps of:
(i) adding the first polynucleotide to the target oligonucleotide in the sample;
(ii) bringing a capture probe for capturing the target oligonucleotide into contact with the target oligonucleotide,
the capture probe containing
(A) a nucleic acid probe,
(B) a solid phase, or an adapter or a linker, adjacent to a nucleotide at the 3′ end or 5′ end of the nucleic acid probe,
the nucleic acid probe containing a sequence complementary to a complete or partial sequence of the target oligonucleotide and/or a complete or partial sequence of the first polynucleotide;
(iii) bringing a single-strand-specific nuclease into contact with the sample subjected to the step of adding the first polynucleotide and the contact step with the capture probe;
(iv) making the capture probe in the sample single-stranded after the contact step with the single-strand-specific nuclease is performed;
(v) bringing a pair of probes capable of self-aggregation composed of first and second oligonucleotides into contact with the sample subjected to the step of making the capture probe single-stranded to form a complex of the capture probe and an oligonucleotide polymer generated by self-aggregation of the first and second oligonucleotides; and
(vi) detecting the complex, and thereby detecting the target oligonucleotide in the sample.

### Claim 5 (depends on 4)

The method of detecting a target oligonucleotide according to claim 4, wherein
(i) the step of adding the first polynucleotide to the target oligonucleotide in the sample is a step of adding the first polynucleotide to the target oligonucleotide in the sample before the contact step with the capture probe.

### Claim 6 (depends on 4)

The method of detecting a target oligonucleotide according to claim 4, wherein
(i) the step of adding the first polynucleotide to the target oligonucleotide in the sample is a step of adding the first polynucleotide to the target oligonucleotide in the sample after the contact step with the capture probe.

### Claim 7 (depends on 4)

The method of detecting a target oligonucleotide according to claim 4, wherein
at least one oligonucleotide of the pair of probes capable of self-aggregation composed of the first and second oligonucleotides contains the complete or partial sequence of the first polynucleotide.

### Claim 8 (depends on 4)

The method of detecting a target oligonucleotide according to claim 4, wherein
the step of forming a complex of (v) includes a step of bringing into contact with an assist probe, and wherein
the assist probe contains the complete or partial sequence of the target oligonucleotide and/or the complete or partial sequence of the first polynucleotide, and a sequence complementary to a complete or partial sequence of at least one of the first and second oligonucleotides.

### Claim 9 (depends on 4)

The method of detecting a target oligonucleotide according to claim 4, wherein the first polynucleotide has a poly(A) sequence, a poly(T) sequence, a poly(U) sequence, a poly(T/U) sequence, a poly(G) sequence, or a poly(C) sequence.

### Claim 10 (depends on 9)

The method of detecting a target oligonucleotide according to claim 9, wherein the first polynucleotide has a poly(A) sequence.

### Claim 11 (depends on 1)

The method of detecting a target oligonucleotide according to claim 1, wherein the first oligonucleotide is an oligonucleotide containing at least a nucleic acid region X, a nucleic acid region Y, and a nucleic acid region Z in order from the 5′-end side, while the second oligonucleotide is an oligonucleotide containing at least a nucleic acid region X′ complementary to the nucleic acid region X, a nucleic acid region Y′ complementary to the nucleic acid region Y, and a nucleic acid region Z′ complementary to the nucleic acid region Z in order from the 5′-end side.

### Claim 12 (depends on 11)

The method of detecting a target oligonucleotide according to claim 11, wherein the nucleic acid region Z contains a poly(A) sequence, and wherein the nucleic acid region Z′ contains a poly(T) sequence.

### Claim 13 (depends on 1)

The method of detecting a target oligonucleotide according to claim 1, wherein the single-strand-specific nuclease is an endonuclease.