# US 2020/0399633 A1 · Oligonucleotide-tethered nucleotides

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- Number: US 2020/0399633 A1
- Document ID: US20200399633A1
- Status: Published application
- Kind: A1
- Application number: 16/908,567
- Filed: 2020-06-22
- Published: 2020-12-24
- Earliest claimed: 2019-06-21
- Assignees: Thermo Fisher Scientific Baltics UAB; Life Technologies Corporation
- Applicants: Thermo Fisher Scientific Baltics Uab; Life Technologies Corporation
- Inventors: Arvydas LUBYS; Inga CIKOTIENE; Zana KAPUSTINA; Arturas BEREZNIAKOVAS; Justina MEDZIUNE; Simona ZEIMYTE; Mark ANDERSEN; Michael ALLEN; Sihong CHEN
- Web page: https://patentgrep.com/patent/US20200399633A1

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- 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}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 2310/3517: Marker; Tag
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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 disclosure describes oligonucleotide-tethered nucleotides, methods of making them, and methods of using them. The oligonucleotide-tethered nucleotides comprise, in some embodiments, a nucleotide linked to an oligonucleotide of from about 3 to about 100 nucleotides in length. These oligonucleotide-tethered nucleotides can be used to label a plurality of different types of nucleic acids in a plurality of different situations with a known oligonucleotide, which can serve as a barcode in some embodiments. The resulting oligonucleotide-labeled nucleic acids oligonucleotides can be used in a variety of nucleic acid sequencing methods.

## Claims

### Claim 1

A method for tagging a nucleic acid with an oligonucleotide comprising:
a. providing the nucleic acid to be tagged,
b. contacting the nucleic acid with a polymerase and at least one oligonucleotide-tethered nucleotide of Formula (A):
or a salt thereof
wherein NB is a nucleobase;
Oligo is an oligonucleotide of 3 to 100 nucleotides;
each of X and Q are independently chosen from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;
each of Z and Y are independently chosen from a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; and
CXN is chosen from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom(s) or a combination thereof where heterocyclic group is optionally substituted at carbon, nitrogen or sulfur atom(s), thereby producing the first tagged nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the contacting comprises contacting the nucleic acid with at least one oligonucleotide-tethered nucleotide, at least one nucleotide not tethered to an oligonucleotide, and a polymerase.

### Claim 3 (depends on 1)

The method of claim 1 further comprising annealing a primer to the nucleic acid.

### Claim 4 (depends on 1)

The method of claim 1, wherein the oligonucleotide-tethered nucleotide is a dideoxynucleotide, optionally wherein the dideoxynucleotide is chosen from dideoxyadenosine triphosphate, dideoxyguanosine triphosphate, dideoxythymidine triphosphate, dideoxyuridine triphosphate, dideoxycytidine triphosphate, and any combination thereof.

### Claim 5 (depends on 1)

The method of claim 1, further comprising:
a. annealing a primer which is at least partially complementary to the tethered oligonucleotide after producing a first tagged nucleic acid strand;
b. contacting the first tagged nucleic acid strand and annealed primer with a nucleic acid polymerase and at least one nucleotide not tethered to an oligonucleotide; and
c. allowing a polymerase to extend from a 3′ hydroxyl of the primer annealed to the tethered oligonucleotide to form a second nucleic acid strand.

### Claim 6 (depends on 1)

The method of claim 1, further comprising:
a. annealing a splint oligonucleotide which is at least partially complementary to the tethered oligonucleotide after producing a first tagged nucleic acid strand;
b. contacting the first tagged nucleic acid strand and annealed splint oligonucleotide with a nucleic acid polymerase and at least one nucleotide not tethered to an oligonucleotide; and
c. allowing a polymerase to extend across the splint oligonucleotide from the 3′ hydroxyl of the tethered oligonucleotide.

### Claim 7

A method for generating a library of nucleic acids from a sample comprising one or more nucleic acids, optionally wherein the sample comprises a plurality of cells, comprising:
a. annealing a first primer which is at least partially complementary to the one or more nucleic acids,
b. contacting the one or more nucleic acids with a nucleic acid polymerase, at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide to form a plurality of nucleic acid strands comprising the oligonucleotide-tethered dideoxynucleotide at their 3′ end;
c. annealing a second primer which is at least partially complementary to the tethered oligonucleotide, and
d. allowing the polymerase to extend from a 3′ hydroxyl of the second primer annealed to the tethered oligonucleotide, thereby producing a library of nucleic acids.

### Claim 8

A method for generating a library of nucleic acids from a sample comprising one or more nucleic acids, optionally wherein the sample comprises a plurality of cells or cell nuclei, comprising:
a. annealing a first primer which is at least partially complementary to the one or more nucleic acids,
b. contacting the one or more nucleic acids with a first nucleic acid polymerase, at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide to form a plurality of first extension products comprising the oligonucleotide-tethered dideoxynucleotide at the 3′ end;
c. annealing a splint oligonucleotide which is at least partially complementary to the tethered oligonucleotide of the first extension products, and
d. contacting the first extension products with a nucleic acid polymerase and one or more nucleotides to allow the polymerase to extend across the annealed splint from the 3′ hydroxyl of the tethered oligonucleotide to produce a second extension product, thereby producing a library of nucleic acids.

### Claim 9 (depends on 8)

The method of claim 8, wherein the one or more nucleic acids in the sample comprises an oligonucleotide of an oligonucleotide-tethered binding agent (OTBA).

### Claim 10 (depends on 9)

The method of claim 9, wherein the tethered oligonucleotide of the OTBA comprises a cell marker binding agent index, and wherein binding agent of the OTBA comprises an aptamer or an antibody or a functional fragment thereof.

### Claim 11 (depends on 8)

The method of claim 8, wherein a sample comprising one or more nucleic acids comprises more than one cell or cell nuclei, wherein the cells or cell nuclei may comprise one or more cell markers, wherein the sample is split into two or more first portions before step a, and wherein each first portion comprises a subpopulation of cells or cell nuclei of the original sample.

### Claim 12 (depends on 11)

The method of claim 11, wherein the first primer comprises a first universal handle sequence and a first barcode, said first barcode being common among the first primers in each first portion, but different from the first barcodes present in first primers in other first portions; and wherein the oligonucleotide of the oligonucleotide-tethered dideoxynucleotide comprises a second universal handle sequence.

### Claim 13 (depends on 12)

The method of claim 12, further comprising, before step c:
a. combining the first portions after formation of the first nucleic extension products and
b. splitting the combined first portions into two or more second portions, wherein the second portions comprise the splint oligonucleotide; wherein the splint oligonucleotide comprises:
i. an oligonucleotide sequence that anneals to the second universal handle on the tethered oligonucleotide;
ii. a template sequence for a second barcode, wherein the second barcodes of each second portion are common, but are different from the second barcodes of other second portions, and
iii. a template sequence for a third universal handle;
wherein the second extension products comprise the second barcode and third universal handle.

### Claim 14 (depends on 13)

The method of claim 13 further comprising:
a. combining the second portions;
b. splitting the combined second portions into two or more third portions;
c. contacting each third portion with amplification primers, wherein the amplification primers are capable of hybridizing to and extending from the first universal handle and the third universal handle, and wherein the amplification primers optionally comprise third and/or fourth barcodes respectively, and first and/or second adapter sequences, respectively, to generate amplification products, wherein the combination of the first, second, and third barcode sequences (or complements thereof) of the amplification products are unique to the amplification products originating from a single cell or nucleus.

### Claim 15 (depends on 7)

The method of claim 7, wherein the primer, the tethered oligonucleotide, or both comprises a random sequence, a target-specific sequence or both.

### Claim 16 (depends on 8)

The method of claim 8, wherein one or more of the primer, the tethered oligonucleotide, or the splint oligonucleotide comprises a universal handle, a universal sequence, a unique molecular identifier, an adapter sequence, a promoter sequence, a barcode sequence, an index sequence, or any combination thereof.

### Claim 17 (depends on 1)

The method of claim 1, wherein the polymerase is a DNA polymerase or an RNA polymerase.

### Claim 18 (depends on 1)

The method of claim 1, wherein the nucleic acid is DNA or RNA.

### Claim 19

An oligonucleotide-tethered nucleotide of Formula (A):
or a salt thereof
wherein NB is a nucleobase;
Oligo is an oligonucleotide of 3 to 100 nucleotides;
each of X and Q are independently chosen from, H, OH, N3, halo, alkyl, alkoxy, alkyl, alkenyl, alkynyl, acyl, cyano, amino, ester, and amido;
each of Z and Y are independently chosen from a bond, amino, amido, alkyl, alkenyl, alkynyl, thioether, sulfonyl, sulfonamido, ether, ketone, carbonyl, anhydride, ester, imido, urea, urethane, and combinations thereof; and
CXN is chosen from alkylene, alkenylene, alkynylene, ketone, carbonate, ester, ether, anhydride, amido, amino, aminoalkylene, imino, imido, diazo, carbamate ester, phosphodiester, sulfide, disulfide, sulfonyl, sulfonamido, and a heterocyclic group containing from one to four N, O, S atom(s) or a combination thereof where heterocyclic group is optionally substituted at carbon, nitrogen or sulfur atom(s).

### Claim 20 (depends on 19)

An oligonucleotide-tethered nucleotide of claim 19, wherein CXN is Click and wherein Click is a product of a click reaction between one of the following pairs of functional groups:
i) alkynyl and azido;
ii) thiol and alkynyl;
iii) thiol and alkenyl;
iv) azido and cyclooctanyl; and
v) cyclooctanyl and nitrone.

### Claim 21

(canceled)