# US 10,480,023 B2 · Method for blocking miRNA

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- Number: US 10,480,023 B2
- Document ID: US10480023B2
- Status: Granted
- Kind: B2
- Application number: 15/565,652
- Filed: 2016-04-10
- Published: 2018-03-15
- Granted: 2019-11-19
- Earliest claimed: 2015-04-10
- Assignees: HudsonAlpha Institute for Biotechnology
- Applicants: HudsonAlpha Institute for Biotechnology
- Inventors: Richard M Myers; Brian S Roberts
- Web page: https://patentgrep.com/patent/US10480023B2

## CPC

- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 2310/113: targeting other non-coding nucleic acids, e.g. antagomirs
- C12N 2320/10: in screening processes
- C12N 2330/31: Libraries, arrays
- C12Q 2521/501: Ligase
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- C12Q 2525/191: incorporating an adaptor
- C12Q 2525/207: siRNA, miRNA
- C12Q 2525/301: Hairpin oligonucleotides
- C12Q 2537/163: blocking probe
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A blocking nucleic acid for use in reducing the abundance of a non-target micro-RNA (miRNA) in an miRNA library is provided, including: a single-stranded complementary region at one of the 5′ end of the blocking nucleic acid or the 3′ end of the blocking nucleic acid, that anneals with a binding region at a first end of the unwanted miRNA; a hairpin loop forming region or other double-stranded region adjacent to the complimentary region, in which all of the terminal ends of the blocking nucleic acid except one are unavailable to participate in ligase reactions. Methods and kits for using the blocking nucleic acid are also provided.

## Claims

### Claim 1

A method of preventing an unwanted micro-RNA (miRNA) that is one of a plurality of miRNAs from participating in reverse transcription polymerase chain reactions (RT-PCR), the unwanted miRNA having a 5′ end and a 3′ end, the method comprising:
(a) annealing a complementary region of a blocking nucleic acid to a binding region at a first end of the unwanted miRNA, wherein the first end of the unwanted miRNA is one of the 5′ end or the 3′ end; and wherein the blocking nucleic acid comprises:
(i) a Crick strand having a 3′ end and a 5′ end;
(ii) a single stranded complementary region at one of the 5′ end of the Crick strand or the 3′ end of the Crick strand, that anneals with the binding region under stringent conditions;
(iii) a double-stranded region on the Crick strand adjacent to the complementary region, the double-stranded region comprising a Watson strand that is annealed to the Crick strand, the Watson strand having a 5′ end and a 3′ end;
(iv) a first blocking moiety linked to the 3′ end of the Crick strand, wherein the first blocking moiety cannot serve as a substrate for ligases;
(v) a second blocking moiety linked to the 5′ end of the Crick strand, wherein the second blocking moiety cannot serve as a substrate for ligases;
(vi) a third blocking moiety linked to the 3′ end of the Watson strand if the complementary region is at the 3′ end of the Crick strand, or linked to the 5′ end of the Watson strand if the complementary region is at the 5′ end of the Crick strand, wherein the third blocking moiety cannot serve as a substrate for ligases; and
(vii) a ligative terminal end on the Watson strand, the ligative terminal end located at the 3′ end of the Watson strand if the complementary region is at the 5′ end of the Crick strand, or at the 5′ end of the Watson strand if the complementary region is at the 3′ end of the Crick strand;
(b) ligating an adenylated nucleic acid adapter to the plurality of miRNAs before or after step (a); and
(c) performing RT-PCR on the plurality of miRNAs.

### Claim 2 (depends on 1)

The method of claim 1, wherein annealing is conducted under stringent conditions.

### Claim 3 (depends on 1)

The method of claim 1, wherein annealing is conducted under maximally stringent conditions.

### Claim 4 (depends on 1)

The method of claim 1, comprising ligating the blocking nucleic acid to the first end of the unwanted miRNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first end of the unwanted miRNA is the 5′ end and the complementary region is at the 5′ end of the blocking nucleic acid.

### Claim 6 (depends on 1)

The method of claim 1, wherein the first end of the unwanted miRNA is the 3′ end and the complementary region is at the 3′ end of the blocking nucleic acid.

### Claim 7 (depends on 1)

The method of claim 1, comprising ligating the blocking nucleic acid to the first end of the unwanted miRNA, wherein the ligating step is performed using a DNA/RNA ligase.

### Claim 8 (depends on 1)

The method of claim 1, wherein the unwanted miRNA is selected from the group consisting of: mir-16, mir-15a, mir-15b, mir-195, mir-424, mir-497, mir-486, mir-451, and mir-26.

### Claim 9 (depends on 1)

A blocked micro RNA (miRNA) complex that is the product of the method of claim 1.

### Claim 10 (depends on 1)

The method of claim 1, wherein an miRNA library with reduced abundance of unwanted miRNA is produced by step (c).

### Claim 11 (depends on 1)

The method of claim 1, comprising introducing an RNA adapter and an RNA ligase under conditions to allow the RNA adapter to ligate to the plurality of miRNAs prior to step (c), and wherein the RNA adapted is blocked from ligating to the unwanted miRNA.