# US 11,441,175 B2 · Methods and kits for depleting undesired nucleic acids > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,441,175 B2 - Document ID: US11441175B2 - Status: Granted - Kind: B2 - Application number: 15/905,952 - Filed: 2018-02-27 - Published: 2018-09-13 - Granted: 2022-09-13 - Earliest claimed: 2017-02-27 - Assignees: Bioo Scientific Corporation - Applicants: BIOO Scientific Corporation - Inventors: Suk Ho Eun; Adam Morris - Web page: https://patentgrep.com/patent/US11441175B2 ## CPC - C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules - C12Q 1/6832: Enhancement of hybridisation reaction - C12Q 1/6855: Ligating adaptors - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 1/6888: for detection or identification of organisms - C12Q 2600/16: Primer sets for multiplex assays - 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 Methods and kits for depleting amplicons that correspond to undesired RNA species present in a sample are provided. The disclosed methods and kits employ a blocker that anneals with at least a portion of the undesired RNA, resulting in a duplex that is not a suitable substrate for ligating an adapter to the end of the undesired RNA. ## Claims ### Claim 1 A method for depleting at least one undesired RNA species having a 5′ end of the undesired RNA and a 3′ end of the undesired RNA, comprising: combining a sample with at least one single-stranded nucleic acid blocker having a 5′ end of the blocker and a 3′ end of the blocker, at least one 3′ adapter, and at least one first ligase to form a first reaction composition and incubating under conditions suitable for first ligation products to be formed, wherein the single-stranded nucleic acid blocker is added before, simultaneously with, or after the 3′ adapter, and wherein the first reaction composition is incubated under conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA to form an RNA-blocker duplex, wherein the RNA-blocker duplex has a blunt duplex end comprising the 5′ end of the undesired RNA and the 3′ end of the blocker, or wherein the 3′ end of the blocker extends beyond the 5′ end of the undesired RNA in the RNA-blocker duplex, such that the 3′ end of the blocker is not present in the RNA-blocker duplex, wherein the RNA-blocker duplex is formed before, simultaneously with, or after the first ligation products are formed, and wherein the blocker does not form a unimolecular duplex under the conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA to form an RNA-blocker duplex; combining the first reaction composition comprising the first ligation products with at least one 5′ adapter and at least one second ligase to form a second reaction composition and incubating under conditions suitable for second ligation products to be formed; wherein neither the 5′ end of the blocker nor the 3′ end of the blocker is ligated to the undesired RNA; and amplifying the second ligation products to generate a pool of amplification products, wherein amplification products corresponding to the at least one undesired RNA species are depleted. ### Claim 2 (depends on 1) The method of claim 1, wherein the amplifying comprises reverse transcription-polymerase chain reaction (RT-PCR). ### Claim 3 (depends on 1) The method of claim 1, wherein the first ligase comprises T4 RNA ligase 2 or truncated T4 RNA ligase 2 and the second ligase comprises T4 RNA ligase 1 or Methanobacterium thermoautotrophicum RNA ligase. ### Claim 4 (depends on 1) The method of claim 1, wherein the first ligase comprises T4 RNA ligase 1, and wherein the first reaction further comprises ATP. ### Claim 5 (depends on 1) The method of claim 1, wherein the single-stranded nucleic acid blocker comprises at least one deoxyribonucleotide, at least one ribonucleotide, at least one locked nucleic acid (LNA), at least one 2′-O-methyl nucleotide, at least one deoxyuridine, at least one inosine, or combinations thereof. ### Claim 6 (depends on 1) The method of claim 1, wherein the single-stranded nucleic acid blocker is shorter or the same length as the undesired RNA to which it anneals. ### Claim 7 (depends on 1) The method of claim 1, wherein the sample comprises at least one small RNA species. ### Claim 8 (depends on 1) The method of claim 1, wherein the amplifying comprises adding at least one oligonucleotide primer and at least one reverse transcriptase to the second ligation products to generate double-stranded second ligation products. ### Claim 9 (depends on 1) The method of claim 1, further comprising separating at least some of the amplification products by size. ### Claim 10 (depends on 1) The method of claim 1, wherein at least one 3′ adapter comprises an activated adenylation (rApp) at its 5′ end, a dideoxy nucleotide at its 3′ end, or an rApp at its 5′ end and a dideoxy nucleotide at its 3′ end. ### Claim 11 A method for depleting at least one undesired RNA species having a 5′ end of the undesired RNA and a 3′ end of the undesired RNA, comprising: combining a sample with at least one single-stranded nucleic acid blocker having a 5′ end of the blocker and a 3′ end of the blocker, at least one 3′ adapter, and at least one first ligase to form a first reaction composition and incubating under conditions suitable for first ligation products to be formed, wherein the single-stranded nucleic acid blocker is added before, simultaneously with, or after the 3′ adapter, and wherein the first reaction composition is incubated under conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA to form an RNA-blocker duplex, wherein the RNA-blocker duplex has a blunt duplex end comprising the 5′ end of the undesired RNA and the 3′ end of the blocker, or wherein the blocker extends beyond the 5′ end of the undesired RNA in the RNA-blocker duplex, wherein the RNA-blocker duplex is formed before, simultaneously with, or after the first ligation products are formed, and wherein the blocker does not form a unimolecular duplex under the conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA to form an RNA-blocker duplex; separating the first ligation products from the first reaction composition; combining the separated first ligation products with at least one 5′ adapter and at least one second ligase to form a second reaction composition and incubating under conditions suitable for second ligation products to be formed; wherein neither the 5′ end of the blocker nor the 3′ end of the blocker is ligated to the undesired RNA; and amplifying the second ligation products to generate a pool of amplification products, wherein amplification products corresponding to the at least one undesired RNA species are depleted. ### Claim 12 (depends on 11) The method of claim 11, wherein the amplifying comprises RT-PCR. ### Claim 13 (depends on 11) The method of claim 11, wherein the first ligase comprises T4 RNA ligase 2 or truncated T4 RNA ligase 2 and the second ligase comprises T4 RNA ligase 1 or Methanobacterium thermoautotrophicum RNA ligase. ### Claim 14 (depends on 11) The method of claim 11, wherein the first ligase comprises T4 RNA ligase 1, and wherein the first reaction further comprises ATP. ### Claim 15 (depends on 11) The method of claim 11, wherein the single-stranded nucleic acid blocker comprises at least one deoxyribonucleotide, at least one ribonucleotide, at least one LNA, at least one 2′-O-methyl nucleotide, at least one deoxyuridine, at least one inosine, or combinations thereof. ### Claim 16 (depends on 11) The method of claim 11, wherein the single-stranded nucleic acid blocker is shorter or the same length as the undesired RNA to which it anneals. ### Claim 17 (depends on 11) The method of claim 11, wherein the sample comprises at least one small RNA species. ### Claim 18 (depends on 11) The method of claim 11, wherein the amplifying comprises adding at least one oligonucleotide primer and at least one reverse transcriptase to the second ligation products to generate double-stranded second ligation products. ### Claim 19 (depends on 11) The method of claim 11, further comprising separating at least some of the amplification products by size. ### Claim 20 (depends on 11) The method of claim 11, wherein at least one 3′ adapter comprises an rApp at its 5′ end, a dideoxy nucleotide at its 3′ end, or an rApp at its 5′ end and a dideoxy nucleotide at its 3′ end. ### Claim 21 A method for depleting at least one undesired RNA species having a 5′ end of the undesired RNA and a 3′ end of the undesired RNA, comprising: combining a sample with at least one 3′ adapter and at least one first ligase to form a first reaction composition and incubating under conditions suitable for first ligation products to be formed; combining the first reaction composition comprising the first ligation products with at least one 5′ adapter, at least one single-stranded nucleic acid blocker having a 5′ end of the blocker and a 3′ end of the blocker, and at least one second ligase to form a second reaction composition and incubating under conditions suitable for second ligation products to be formed, wherein the at least one single-stranded nucleic acid blocker is added before, simultaneously with, or after the 5′ adapter, and wherein the second reaction composition is incubated under conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA or first ligation products comprising undesired RNA to form an RNA-blocker duplex, wherein the RNA-blocker duplex has a blunt duplex end comprising the 5′ end of the undesired RNA and the 3′ end of the blocker, or wherein the blocker extends, in single-stranded form, beyond the 5′ end of the undesired RNA in the RNA-blocker duplex, wherein the RNA-blocker duplex is formed before or simultaneously with the forming of the second ligation products; and amplifying the second ligation products to generate a pool of amplification products, wherein amplification products corresponding to the at least one undesired RNA species are depleted. ### Claim 22 (depends on 21) The method of claim 21, wherein the amplifying comprises RT-PCR. ### Claim 23 (depends on 21) The method of claim 21, wherein the first ligase comprises T4 RNA ligase 2 or truncated T4 RNA ligase 2 and the second ligase comprises T4 RNA ligase 1 or Methanobacterium thermoautotrophicum RNA ligase. ### Claim 24 (depends on 21) The method of claim 21, wherein the first ligase comprises T4 RNA ligase 1, and wherein the first reaction further comprises ATP. ### Claim 25 (depends on 21) The method of claim 21, wherein the single-stranded nucleic acid blocker comprises at least one deoxyribonucleotide, at least one ribonucleotide, at least one LNA, at least one 2′-O-methyl nucleotide, at least one deoxyuridine, at least one inosine, or combinations thereof. ### Claim 26 (depends on 21) The method of claim 21, wherein the single-stranded nucleic acid blocker is shorter or the same length as the undesired RNA to which it anneals. ### Claim 27 (depends on 21) The method of claim 21, wherein the sample comprises at least one small RNA species. ### Claim 28 (depends on 21) The method of claim 21, wherein the amplifying comprises adding at least one oligonucleotide primer and at least one reverse transcriptase to the second ligation products to generate double-stranded second ligation products. ### Claim 29 (depends on 21) The method of claim 21, further comprising separating at least some of the amplification products by size. ### Claim 30 (depends on 21) The method of claim 21, wherein at least one 3′ adapter comprises an rApp at its 5′ end, a dideoxy nucleotide at its 3′ end, or an rApp at its 5′ end and a dideoxy nucleotide at its 3′ end. ### Claim 31 A method for depleting at least one undesired RNA species having a 5′ end of the undesired RNA and a 3′ end of the undesired RNA, comprising: combining a sample with at least one 3′ adapter and at least one first ligase to form a first reaction composition and incubating under conditions suitable for first ligation products to be formed; separating the first ligation products from the first reaction composition; combining the separated first ligation products with at least one 5′ adapter, at least one single-stranded nucleic acid blocker having a 5′ end of the blocker and a 3′ end of the blocker, and at least one second ligase to form a second reaction composition and incubating under conditions suitable for second ligation products to be formed, wherein the at least one single-stranded nucleic acid blocker is added before, simultaneously with, or after the 5′ adapter, and wherein the second reaction composition is incubated under conditions suitable for the single-stranded nucleic acid blocker to anneal with the undesired RNA or first ligation products comprising undesired RNA to form an RNA-blocker duplex, wherein the RNA-blocker duplex has a blunt duplex end comprising the 5″ end of the undesired RNA and the 3′ end of the blocker, or wherein the blocker extends, in single-stranded form, beyond the 5′ end of the undesired RNA in the RNA-blocker duplex, wherein the RNA-blocker duplex is formed before or simultaneously with the forming of the second ligation products; and amplifying the second ligation products to generate a pool of amplification products, wherein amplification products corresponding to the at least one undesired RNA species are depleted. ### Claim 32 (depends on 31) The method of claim 31, wherein the amplifying comprises RT-PCR. ### Claim 33 (depends on 31) The method of claim 31, wherein the first ligase comprises T4 RNA ligase 2 or truncated T4 RNA ligase 2 and the second ligase comprises T4 RNA ligase 1 or Methanobacterium thermoautotrophicum RNA ligase. ### Claim 34 (depends on 31) The method of claim 31, wherein the first ligase comprises T4 RNA ligase 1, and wherein the first reaction further comprises ATP. ### Claim 35 (depends on 31) The method of claim 31, wherein the single-stranded nucleic acid blocker comprises at least one deoxyribonucleotide, at least one ribonucleotide, at least one LNA, at least one 2′-O-methyl nucleotide, at least one deoxyuridine, at least one inosine, or combinations thereof. ### Claim 36 (depends on 31) The method of claim 31, wherein the single-stranded nucleic acid blocker is shorter or the same length as the undesired RNA to which it anneals. ### Claim 37 (depends on 31) The method of claim 31, wherein the sample comprises at least one small RNA species. ### Claim 38 (depends on 31) The method of claim 31, wherein the amplifying comprises adding at least one oligonucleotide primer and at least one reverse transcriptase to the second ligation products to generate double-stranded second ligation products. ### Claim 39 (depends on 31) The method of claim 31, further comprising separating at least some of the amplification products by size. ### Claim 40 (depends on 31) The method of claim 31, wherein at least one 3′ adapter comprises an rApp at its 5′ end, a dideoxy nucleotide at its 3′ end, or rApp at its 5′ end and a dideoxy nucleotide at its 3′ end.