# US 11,898,195 B2 · Methods and compositions for determining pH

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- Number: US 11,898,195 B2
- Document ID: US11898195B2
- Status: Granted
- Kind: B2
- Application number: 17/466,474
- Filed: 2021-09-03
- Published: 2021-12-30
- Granted: 2024-02-13
- Earliest claimed: 2015-05-19
- Assignees: The University of Chicago; National Centre for Biological Sciences
- Applicants: The University of Chicago; National Centre for Biological Sciences
- Inventors: Yamuna Krishnan; Saheli Halder
- Web page: https://patentgrep.com/patent/US11898195B2

## CPC

- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12Q 1/005: {involving specific analytes or enzymes (including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence)}
- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G01N 33/84: involving inorganic compounds or pH
- A01K 2207/05: Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
- A01K 2227/703: Worms, e.g. Caenorhabdities elegans
- A01K 2267/0306: Animal model for genetic diseases
- A01K 2267/0362: Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/3517: Marker; Tag
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

Described herein are nucleic acid molecules and complexes useful as i-switch pH reporters that have increased sensitivities as a pH reporter and have alternate pH reporting capacity ranges. Aspects of the disclosure relate to a method for determining pH comprising providing a nucleic acid complex comprising: a first single-stranded nucleic acid molecule comprising the sequence CnXCnYCnZCn (SEQ ID NO. 6) wherein C is cytosine; X, Y and Z are each one or more of adenine, thymine, guanine, or combinations thereof; and n is greater than or equal to 2; and wherein at least 2 cytosine residues of the first single-stranded nucleic acid molecule are modified; and a second single-stranded nucleic acid molecule that is partially or fully complementary to the first single-stranded molecule, wherein a first label is conjugated to the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule; and wherein the first label is capable of producing a signal, wherein the intensity of the signal varies as a function of the conformation of the nucleic acid complex; and measuring the intensity of the signal and determining the pH from the measured signal.

## Claims

### Claim 1

A nucleic acid complex comprising:
i) a first single-stranded nucleic acid molecule comprising the nucleic acid sequence:
(SEQ ID NO. 8)
5′-CCCTAACCCCTAAC*CC*CTAACC*CC*ATATATATCCTAGAACGAC
AGACAAACAGTGAGTC-3;
(SEQ ID NO. 9)
5′-CCC*CTAACC*CCTAACCC*CTAACC*CCATATATATCCTAGAACGA
CAGACAAACAGTGAGTC-3′;
(SEQ ID NO. 10)
5′-C*CCCTAACCCC*TAAC*CCCTAACCCC*ATATATATCCTAGAACGA
CAGACAAACAGTGAGTC-3′;
or
(SEQ ID NO. 11)
5′-C*C*C*C*TAACCCCTAACCCCTAACCCCATATATATCCTAGAACGA
CAGACAAACAGTGAGTC-3′;
wherein C* is a 5′-bromocytosine or 5′-methylcytosine and wherein the first single-stranded nucleic acid molecule is conjugated to a first fluorophore which comprises a donor fluorophore as a first label; and
ii) a second single-stranded nucleic acid molecule that is partially or fully complementary to the first single-stranded molecule, wherein the second single-stranded nucleic acid molecule comprises the nucleic acid sequence:
5′-GACTCACTGTTTGTCTGTCGTTCTAGGATAT*ATATTTTGTTATGTGTTATGTGTTA T-3′ (SEQ ID NO:19)(P comp) and wherein T* is conjugated to a second fluorophore which comprises an acceptor fluorophore as a second label,
wherein the first and the second labels arc a donor-acceptor pair.

### Claim 2 (depends on 1)

The nucleic acid complex of claim 1, wherein C* is 5′-bromocysteine.

### Claim 3 (depends on 1)

The nucleic acid complex of claim 1, wherein C* is 5′-methylcytosine.