# US 10,246,706 B2 · Methods and compositions for preventing concatemerization during template-switching

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- Number: US 10,246,706 B2
- Document ID: US10246706B2
- Status: Granted
- Kind: B2
- Application number: 15/947,971
- Filed: 2018-04-09
- Published: 2018-08-09
- Granted: 2019-04-02
- Earliest claimed: 2016-08-03
- Assignees: New England Biolabs, Inc.
- Applicants: New England Biolabs, Inc.
- Inventors: Shengxi Guan; Thomas C. Evans, Jr.; Nicole Nichols; Yanxia Bei
- Web page: https://patentgrep.com/patent/US10246706B2

## CPC

- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Y 207/07049: RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
- 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
- C12Y: ENZYMES

## Abstract

Compositions and methods for performing a template-switching reaction are provided that may include reducing or eliminating concatemerization of the template-switching oligonucleotide (TSO). In some embodiments, the composition may comprise: a reverse transcriptase; a TSO that includes a recognition sequence for a site-specific double strand nucleic acid cleaving enzyme, wherein the TSO has at its 3′ end at least one nucleotide capable of hybridizing to at least one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and a site-specific double strand nucleic acid cleaving enzyme that cleaves the TSO at the recognition sequence.

## Claims

### Claim 1

A method for making cDNA comprising:
(a) obtaining a reaction mix comprising:
a reverse transcriptase;
an RNA template;
a cDNA synthesis primer;
an oligonucleotide for template switching, the oligonucleotide comprising a 3′ end having at least one nucleotide that can hybridize to one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and
polyethylene glycol having an average molecular weight in the range of about 3350 to about 8000; and
(b) incubating the reaction mix so as to produce cDNA molecules that comprise, at the 3′ end of the cDNA, an added sequence comprising the complement of a sequence at the 3′ end of the template switching oligonucleotide.

### Claim 2 (depends on 1)

The method of claim 1, wherein (b) further comprises incubating the reaction mix with a site-specific double stranded nucleic acid cleaving enzyme for preventing concatamers of the double stranded form of the template-switching oligonucleotide at the 3′ end of the cDNA molecules.

### Claim 3 (depends on 1)

The method of claim 1, wherein step (b) comprises incubating the reaction mix at a temperature of 35° C. to 55° C. for a period of at least 30 minutes.

### Claim 4 (depends on 2)

The method of claim 2, further comprising, after step (b), inactivating the reverse transcriptase and the site-specific nucleic acid cleaving enzyme by incubating the reaction mix at a temperature of at least 65° C. for a period of at least 10 minutes.

### Claim 5 (depends on 1)

The method of claim 1, further comprising: (c) amplifying the cDNA molecules to produce amplification products.

### Claim 6 (depends on 5)

The method of claim 5, wherein (c) is performed using a first primer and a second primer, wherein the first primer hybridizes to the added sequence at the 3′ end of the cDNA and the second primer hybridizes to the complement of a sequence in the cDNA synthesis primer; and wherein the amplification is performed by PCR.

### Claim 7 (depends on 1)

A method according to claim 1, further comprising obtaining an enhanced yield of amplified cDNA in the presence of the crowding agent compared to in the absence of the crowding agent.

### Claim 8 (depends on 7)

The method of claim 7, wherein the method further comprises sequencing the amplification products.

### Claim 9 (depends on 1)

The method of claim 1, wherein the RNA template is from a clinical sample.

### Claim 10

A composition comprising:
(a) a reverse transcriptase;
(b) an oligonucleotide for template switching, wherein the oligonucleotide comprises a 3′ end having at least one nucleotide that can hybridize to one or more non-templated nucleotides added to a templated cDNA strand by the reverse transcriptase; and
(c) polyethylene glycol having an average molecular weight in the range of about 3350 to about 8000.

### Claim 11 (depends on 10)

The composition according to claim 10, wherein the template-switching oligonucleotide comprises a recognition sequence for a site-specific double stranded strand nucleic acid cleaving enzyme; and wherein the reaction mix further comprises a site-specific double strand nucleic acid cleaving enzyme that recognizes the recognition sequence in the template-switching oligonucleotide.

### Claim 12 (depends on 10)

The composition of claim 10, further comprising an RNA template.

### Claim 13 (depends on 11)

The composition of claim 11, wherein the RNA template is total RNA or polyA±RNA.

### Claim 14 (depends on 11)

The composition of claim 11, wherein the RNA template comprises a 5′ modified GMP cap.

### Claim 15 (depends on 13)

The composition of claim 13, wherein the 5′ modified GMP cap is a 7-methylguanosine or an affinity tag-labeled GMP cap.

### Claim 16 (depends on 10)

The composition of claim 10, further comprising a cDNA synthesis primer which is an oligo(dT) primer, a random primer, or a transcript-specific primer.

### Claim 17 (depends on 12)

The composition of claim 12, further comprising a cDNA synthesis primer having a 5′ tail that does not hybridize to the RNA template.

### Claim 18 (depends on 10)

The composition of claim 10, wherein the template-switching oligonucleotide is a DNA-RNA chimera.

### Claim 19 (depends on 10)

The composition of claim 10, wherein the template-switching oligonucleotide comprises an amplification primer sequence, and optionally a molecular barcode sequence.

### Claim 20 (depends on 10)

The composition of claim 10, wherein the template-switching oligonucleotide comprises three riboguanine residues at its 3′ end.

### Claim 21 (depends on 10)

The composition of claim 10, wherein the reverse transcriptase is Moloney murine leukemia virus (M-MLV), M-MLV reverse transcriptase lacking RNaseH activity, human T-cell leukemia virus type I (HTLV-I) reverse transcriptase, bovine leukemia virus (BLV) reverse transcriptase, Rous sarcoma virus (RSV) reverse transcriptase, or human immunodeficiency virus (HIV) reverse transcriptase, or a variant thereof.

### Claim 22 (depends on 11)

The composition of claim 11, wherein the site-specific double strand nucleic acid cleaving enzyme is selected from a restriction endonuclease, an N-glycosylase, a lyase (N-glycosylase-lyase, an endonuclease, a cas9 ortholog and an Argonaut ortholog.

### Claim 23 (depends on 11)

The composition of claim 11, wherein the restriction endonuclease is active at a temperature in the range of 40° C. to 55° C.

### Claim 24 (depends on 22)

The composition of claim 22, wherein the restriction endonuclease is capable of inactivation by incubation at a temperature of at least 65° C. for at least 10 minutes.