# US 2020/0399639 A1 · Detection of microbial endotoxins in oral samples using aptamers

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- Number: US 2020/0399639 A1
- Document ID: US20200399639A1
- Status: Published application
- Kind: A1
- Application number: 16/867,587
- Filed: 2020-05-06
- Published: 2020-12-24
- Earliest claimed: 2019-05-06
- Assignees: The Procter & Gamble Company
- Applicants: The Procter & Gamble Company
- Inventors: Sancai Xie; Thomas Glenn Huggins, JR.; Cheryl Sue Tansky; Ping Hu; Susan Ellen Forest
- Web page: https://patentgrep.com/patent/US20200399639A1

## CPC

- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/56911: {Bacteria}
- C12N 2310/16: Aptamers
- C12N 2310/317: with an inverted bond, e.g. a cap structure
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N 2310/351: Conjugate
- C12N 2310/3513: Protein; Peptide
- G01N 2400/50: Lipopolysaccharides; LPS
- G01N 2800/18: Dental and oral disorders
- 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)

## Abstract

The present invention is directed to an aptamer composition comprising at least one oligonucleotide consisting of: deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, and mixtures thereof; wherein said aptamer composition has a binding affinity for one or more bacterial species from the genera Prevotella and Porphyromonas.

## Claims

### Claim 1

An aptamer composition comprising an oligonucleotide that is at least one of deoxyribonucleotides, ribonucleotides, derivatives of deoxyribonucleotides, derivatives of ribonucleotides, or mixtures thereof;
wherein said aptamer composition has a binding affinity for one or more bacterial species from the genus Prevotella or the genus Porphyromonas.

### Claim 2 (depends on 1)

The aptamer composition of claim 1, wherein said one or more Prevotella species are at least one of: Prevotella baroniae, Prevotella bivia, Prevotella buccae, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella loescheii, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pollens, Prevotella pleuritidis, Prevotella saccharolytica, Prevotella salivae, Prevotella_sp_C561, Prevotellasp_oral_taxon 306, Prevotellasp_oral_taxon 317, Prevotellasp_oral_taxon 473, Prevotella timonensis, Prevotella veroralis, or mixtures thereof.

### Claim 3 (depends on 1)

The aptamer composition of claim 1, wherein said one or more Prevotella species are at least one of: Prevotella denticola, Prevotella intermedia, Prevotella melaninogenica, Prevotella nigrescens, Prevotella pollens, Prevotella salivae, or mixtures thereof.

### Claim 4 (depends on 1)

The aptamer composition of claim 1, wherein said one or more Porphyromonas species are at least one of: Porphyromonas asaccharolytica, Porphyromonas catoniae, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonassp_oral_taxon 278, Porphyromonassp_oral_taxon 279, Porphyromonas_uenonis, or mixtures thereof.

### Claim 5 (depends on 1)

The aptamer composition of claim 1, wherein said one or more Porphyromonas species are at least one of: Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonassp_oral_taxon 278, or mixtures thereof.

### Claim 6 (depends on 1)

The aptamer composition of claim 1, wherein the oligonucleotide has at least 50% nucleotide sequence identity to SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.

### Claim 7 (depends on 1)

The aptamer composition of claim 1, wherein the oligonucleotide comprises at least one oligonucleotide of SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.

### Claim 8 (depends on 1)

The aptamer composition of claim 1, wherein the oligonucleotide comprises one or more motifs of SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.

### Claim 9 (depends on 1)

The aptamer composition of claim 1, wherein the oligonucleotide comprises natural or non-natural nucleobases.

### Claim 10 (depends on 3)

The aptamer composition of claim 3, wherein said non-natural nucleobases are at least one of hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine, 5-hydroxymethylcyto sine, thiouracil, 1-methylhypoxanthine, 6-methylisoquinoline-1-thione-2-yl, 3-methoxy-2-naphthyl, 5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl, 7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl, phenoxazinyl, phenoxazinyl-G-clam, or mixtures thereof.

### Claim 11 (depends on 1)

The aptamer composition of claim 1, wherein the nucleosides of the oligonucleotide are linked by a chemical motif that is at least one of natural phosphate diester, chiral phosphorothionate, chiral methyl phosphonate, chiral phosphoramidate, chiral phosphate chiral triester, chiral boranophosphate, chiral phosphoroselenoate, phosphorodithioate, phosphorothionate amidate, methylenemethylimino, 3′-amide, 3′ achiral phosphoramidate, 3′ achiral methylene phosphonates, thioformacetal, thioethyl ether, or mixtures thereof.

### Claim 12 (depends on 1)

The aptamer composition of claim 1, where said derivatives of ribonucleotides or said derivatives of deoxyribonucleotides are at least one of locked oligonucleotides, peptide oligonucleotides, glycol oligonucleotides, threose oligonucleotides, hexitol oligonucleotides, altritol oligonucleotides, butyl oligonucleotides, L-ribonucleotides, arabino oligonucleotides, 2′-fluoroarabino oligonucleotides, cyclohexene oligonucleotides, phosphorodiamidate morpholino oligonucleotides, or mixtures thereof.

### Claim 13 (depends on 1)

The aptamer composition of claim 1, further comprising at least one polymeric material, wherein said at least one polymeric material is covalently linked to the oligonucleotide.

### Claim 14 (depends on 13)

The aptamer composition of claim 13, wherein said at least one polymeric material is polyethylene glycol.

### Claim 15 (depends on 1)

The aptamer composition of claim 1, wherein the nucleotides at the 5′- and 3′-ends of the oligonucleotide are inverted.

### Claim 16 (depends on 1)

The aptamer composition of claim 1, wherein at least one nucleotide of the oligonucleotide is fluorinated at the 2′ position of the pentose group.

### Claim 17 (depends on 1)

The aptamer composition of claim 1, wherein the pyrimidine nucleotides of the oligonucleotide are fluorinated at the 2′ position of the pentose group.

### Claim 18 (depends on 1)

The aptamer composition of claim 1, wherein said at least one oligonucleotide is covalently or non-covalently attached to one or more reporter molecules; wherein said one or more reporter molecules are at least one of gold nanoparticles, fluorescent tags, horse radish peroxidase, alkaline phosphatase, green fluorescence proteins and latex, or mixtures thereof.

### Claim 19 (depends on 1)

The aptamer composition of claim 1, wherein said one or more reporter molecules comprise gold nanoparticles.

### Claim 20

A method for detecting endotoxins and outer membrane vesicles comprising:
a) obtaining an oral cavity sample;
b) applying an oral cavity sample to an assay kit; and
c) measuring an assay result.

### Claim 21 (depends on 20)

The method of claim 20, wherein said a sample includes: saliva, oral lavage, subgingival plaques and supragingival plaques.

### Claim 22 (depends on 20)

The method of claim 20, wherein said an assay kit comprises at least one oligonucleotide of SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.

### Claim 23 (depends on 20)

The method of claim 20, wherein said an assay kit comprises at least one oligonucleotide with at least 50% nucleotide sequence identity to SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.

### Claim 24 (depends on 20)

The method of claim 20, wherein said an assay kit comprises at least one oligonucleotide with the oligonucleotide with natural or non-natural nucleobases of SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.

### Claim 25 (depends on 20)

The method of claim 20, wherein the assay kit contains an oligonucleotide that is at least one of SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.

### Claim 26 (depends on 20)

The method of claim 20, wherein said at least one oligonucleotide is covalently or non-covalently attached to one or more reporter molecules; wherein said one or more reporter molecules are at least one of gold nanoparticles, fluorescent tags, horse radish peroxidase, alkaline phosphatase, green fluorescence proteins and latex, or mixtures thereof.

### Claim 27 (depends on 20)

The method of claim 20, wherein the assay kit includes point of care assays and 96-well assays.

### Claim 28 (depends on 27)

The method of claim 27, wherein said point of care assays include lateral flow assays.

### Claim 29 (depends on 26)

The method of claim 26, wherein said point of care assays include competitive and non-competitive lateral flow assay.

### Claim 30 (depends on 28)

The method of claim 28, wherein said point of care assays include oligonucleotides with DNA or RNA sequences complementary to at least one of SEQ ID NO 1001 to SEQ ID NO 1105 or SEQ ID NO 2001 to SEQ ID NO 2108.