# US 12,000,000 B2 · Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing

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- Number: US 12,000,000 B2
- Document ID: US12000000B2
- Status: Granted
- Kind: B2
- Application number: 18/045,436
- Filed: 2022-10-10
- Published: 2023-11-16
- Granted: 2024-06-04
- Earliest claimed: 2015-11-20
- Assignees: Pacific Biosciences of California, Inc.
- Applicants: Pacific Biosciences of California, Inc.
- Inventors: Lubomir Sebo; Gene Shen; Stephen Yue; Honey Osuna; Yuri Lapin; Louis Brogley; Andrei Fedorov
- Web page: https://patentgrep.com/patent/US12000000B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/207: the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C09B 69/105: {containing a methine or polymethine dye}
- 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
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## Abstract

Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

## Claims

### Claim 1

A labeled nucleotide analog comprising:
a first avidin protein having four subunits, each subunit comprising one biotin binding site;
a first nucleotide compound, bound to the first avidin protein, the first nucleotide compound comprising a polyphosphate element, a nucleoside element, an optional multivalent central core element, a terminal coupling element, and a nucleotide linker element; and
a first dye-labeled compound, bound to the first avidin protein;
wherein the first dye-labeled compound is a compound of structural formula (IIIG):
wherein
each L′ is independently a dye compound linker element;
each S is independently a shield element;
each Dye is independently either an acceptor dye or a donor dye;
each B″ is independently a terminal coupling element;
each p is independently 0 or 1; and
each r″ is independently an integer from 0 to 8;
s is an integer from 1 to 6; and
t is 0 or 1;
wherein the compound of structural formula (IIIG) comprises at least one shield element,
at least one acceptor dye, and at least one donor dye; and
wherein the at least one shield element does not comprise a protein.

### Claim 2 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the first dye-labeled compound and the first nucleotide compound are each bound to the first avidin protein through a biotin moiety.

### Claim 3 (depends on 1)

The labeled nucleotide analog of claim 1, further comprising a second avidin protein and a second nucleotide compound, wherein
the second avidin protein is bound to the first dye-labeled compound through a biotin moiety and to the second nucleotide compound through a biotin moiety.

### Claim 4 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the first avidin protein is avidin, streptavidin, tamavidin, traptavidin, xenavidin, bradavidin, AVR2, AVR4, or a homolog thereof.

### Claim 5 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the donor dye and the acceptor dye are fluorescent dyes.

### Claim 6 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the first dye-labeled compound comprises at least two donor dyes.

### Claim 7 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the first dye-labeled compound comprises at least two acceptor dyes.

### Claim 8 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the first dye-labeled compound comprises at least two donor dyes and at least two acceptor dyes.

### Claim 9 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the dye compound linker element comprises a shield element or a side chain element.

### Claim 10 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the shield element of the first dye-labeled compound decreases photodamage of the first dye-labeled compound or of a biomolecule associated with the analog or increases brightness of the analog.

### Claim 11 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the shield element comprises a plurality of side chains.

### Claim 12 (depends on 11)

The labeled nucleotide analog of claim 11, wherein at least one side chain has a molecular weight of at least 300.

### Claim 13 (depends on 11)

The labeled nucleotide analog of claim 11, wherein all of the side chains have a molecular weight of at least 300.

### Claim 14 (depends on 11)

The labeled nucleotide analog of claim 11, wherein at least one side chain comprises a polyethylene glycol.

### Claim 15 (depends on 11)

The labeled nucleotide analog of claim 11, wherein at least one side chain comprises a negatively-charged component.

### Claim 16 (depends on 11)

The labeled nucleotide analog of claim 11, wherein at least one side chain comprises a substituted phenyl group.

### Claim 17 (depends on 11)

The labeled nucleotide analog of claim 11, wherein at least one side chain comprises a triazole.

### Claim 18 (depends on 11)

The labeled nucleotide analog of claim 11, wherein at least one side chain comprises the structure:

### Claim 19 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the shield element comprises the structure:
wherein each y is independently an integer from 1 to 6.

### Claim 20 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the shield element comprises the structure:

### Claim 21 (depends on 1)

The labeled nucleotide analog of claim 1, comprising at least two terminal coupling elements.

### Claim 22 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the terminal coupling element comprises a biotin moiety.

### Claim 23 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the acceptor dye or dyes or the donor dye or dyes is a cyanine dye.

### Claim 24 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the first dye-labeled compound does not contain a nucleoside.

### Claim 25 (depends on 1)

The labeled nucleotide analog of claim 1, wherein each r″ is independently an integer from 0 to 4.

### Claim 26 (depends on 1)

The labeled nucleotide analog of claim 1 comprising at least two donor dyes.

### Claim 27 (depends on 1)

The labeled nucleotide analog of claim 1 comprising at least two acceptor dyes.

### Claim 28 (depends on 1)

The labeled nucleotide analog of claim 1 comprising at least two donor dyes and at least two acceptor dyes.

### Claim 29 (depends on 1)

The labeled nucleotide analog of claim 1, wherein at least one dye compound linker element comprises a shield element or a side chain element.

### Claim 30 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the at least one shield element comprises a plurality of side chains.

### Claim 31 (depends on 30)

The labeled nucleotide analog of claim 30, wherein at least one side chain has a molecular weight of at least 300.

### Claim 32 (depends on 30)

The labeled nucleotide analog of claim 30, wherein all of the side chains have a molecular weight of at least 300.

### Claim 33 (depends on 30)

The labeled nucleotide analog of claim 30, wherein at least one side chain comprises a polyethylene glycol.

### Claim 34 (depends on 30)

The labeled nucleotide analog of claim 30, wherein at least one side chain comprises a negatively-charged component.

### Claim 35 (depends on 30)

The labeled nucleotide analog of claim 30, wherein at least one side chain comprises a substituted phenyl group.

### Claim 36 (depends on 30)

The labeled nucleotide analog of claim 30, wherein at least one side chain comprises a triazole.

### Claim 37 (depends on 30)

The labeled nucleotide analog of claim 30, wherein at least one side chain comprises the structure:

### Claim 38 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the at least one shield element comprises the structure:
wherein each y is independently an integer from 1 to 6.

### Claim 39 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the at least one shield element comprises the structure:

### Claim 40 (depends on 1)

The labeled nucleotide analog of claim 1, comprising two terminal coupling elements.

### Claim 41 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the terminal coupling element comprises a biotin moiety.

### Claim 42 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the at least one acceptor dye or the at least one donor dye is a cyanine dye.

### Claim 43 (depends on 1)

The labeled nucleotide analog of claim 1, wherein the first dye-labeled compound does not contain a nucleoside.