# US 12,760,354 B2 · In-situ concatenation of oligo-nucleotide probes for target detection

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- Number: US 12,760,354 B2
- Document ID: US12760354B2
- Status: Granted
- Kind: B2
- Application number: 17/618,384
- Filed: 2020-06-15
- Published: 2022-10-06
- Granted: 2026-10-13
- Earliest claimed: 2019-06-13
- Assignees: Qbiotix Limited
- Applicants: Qbiotix Limited
- Inventors: Ian Thrippleton
- Web page: https://patentgrep.com/patent/US12760354B2

## CPC

- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- 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 relates to a multiplicity of nucleic acid probes, a composition comprising said probes and uses thereof. The invention relates to a multiplicity of non-beacon, hairpin loop forming nucleic acid probes. In particular, the invention relates to a multiplicity of nucleic acid probes, each nucleic acid probe comprising a nucleic acid sequence complementary to the target nucleic acid sequence; wherein said nucleic acid probes are capable of forming a multimer in-situ with at least a neighbouring probe. The invention relates to the use of nucleic acid probes to detect the presence or absence of organisms in biological samples.

## Claims

### Claim 1

A multiplicity of hairpin loop forming nucleic acid probes capable of concatenating in situ to form a concatenated probe complementary to a target nucleic acid sequence, each nucleic acid probe of the multiplicity comprising:
a nucleic acid sequence complementary to the target nucleic acid sequence;
a 5′ flanking sequence comprising a first detection moiety; and
a 3′ flanking sequence comprising a second detection moiety,
wherein the 5′ flanking sequence and the 3′ flanking sequence of a single nucleic acid probe are at least partially complementary to each other;
wherein the multiplicity of nucleic acid probes comprises at least two populations of probe; a first population characterized in that the first detection moiety and the second detection moiety on a single nucleic acid probe are the same and are each of type A; and second population characterized in that the first detection moiety and the second detection moiety on a single nucleic acid probe are the same and are each of type B;
wherein said multiplicity of nucleic acid probes are capable of concatenating in situ to form a concatenated probe complementary to the target sequence, in which probes of the first population alternate with probes of the second population, such that the 5′ flanking sequence of a first nucleic acid probe of a first population interlinks with at least part of the 3′ flanking sequence of a neighboring nucleic acid probe of the second population to form a stem structure such that the first detection moiety of the first nucleic acid probe and the second detection moiety of the neighboring nucleic acid probe of the stem structure interact to generate a signal.

### Claim 2 (depends on 1)

The multiplicity of nucleic acid probes of claim 1, wherein the 5′ and/or 3′ flanking-sequences do not hybridise with the target nucleic acid sequence.

### Claim 3 (depends on 1)

The multiplicity of nucleic acid probes of claim 1, wherein the stem structure has a significantly less negative ΔG and a Tm-value below the hybridization temperature with respect to the hybridization target.

### Claim 4 (depends on 1)

The multiplicity of nucleic acid probes of claim 1, wherein the probe nucleic acid sequence complementary to the target nucleic acid sequence comprises ribonucleotides, ribonucleotide analogues, deoxyribonucleotides, deoxyribonucleotide analogues, or a combination thereof.

### Claim 5 (depends on 1)

The multiplicity of nucleic acid probes of claim 1, wherein the probe nucleic acid sequence complementary to the target nucleic acid sequence is DNA and the 5′ and 3′ flanking sequences of the probe comprise DNA-analogues.

### Claim 6 (depends on 1)

The multiplicity of nucleic acid probes of claim 1, wherein the target nucleic acid sequence is DNA or RNA.

### Claim 7 (depends on 1)

The multiplicity of nucleic acid probes of claim 1, wherein said first detection moiety of the first nucleic acid probe is an energizer (type-a probe) and said second detection moiety of the neighboring nucleic acid probe is a molecule capable of energy resonance transfer (type-b probe); or wherein said first detection moiety of the first nucleic acid probe is a molecule capable of energy resonance transfer (type-b probe) and said second detection moiety of the neighboring nucleic acid probe is an energizer (type-a probe).