# US 2017/0283797 A1 · Compositions and methods relating to synthetic RNA polynucleotides created from synthetic DNA oligonucleotides

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- Number: US 2017/0283797 A1
- Document ID: US20170283797A1
- Status: Published application
- Kind: A1
- Application number: 15/469,681
- Filed: 2017-03-27
- Published: 2017-10-05
- Earliest claimed: 2016-04-01
- Assignees: New England Biolabs, Inc.
- Applicants: New England Biolabs, Inc.
- Inventors: G. B. Robb; Isaac B. Meek; Dianne S. Schwarz; Ezra Schildkraut
- Web page: https://patentgrep.com/patent/US20170283797A1

## CPC

- C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- 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}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 2310/16: Aptamers
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2320/13: in a process of directed evolution, e.g. SELEX, acquiring a new function
- C12N 2330/00: Production
- C12N 2330/31: Libraries, arrays
- C12Q 1/68: involving nucleic acids
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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

Compositions and methods are provided for forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber using combined reagents in a single step reaction. DNA polymerase, RNA polymerase and single stranded (ss) DNA oligonucleotides are combined where each DNA oligonucleotide has one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and forming a single RNA polynucleotide, excluding the RNA promoter sequence, derived from the first and second DNA oligonucleotides

## Claims

### Claim 1

A method of forming a single RNA polynucleotide from a plurality of DNA oligonucleotides in a single reaction chamber in a single step reaction, comprising:
(a) combining with a DNA polymerase and an RNA polymerase, a first and second synthetic single strand (ss) DNA oligonucleotide, each having one or more sequence modules, wherein one sequence module in the first ss DNA oligonucleotide is complementary to a sequence module at the 3′ end of the second ss DNA oligonucleotide; and wherein a second module on the first ss DNA oligonucleotide is an RNA polymerase promoter sequence; and
(b) forming a single RNA polynucleotide derived from the first and second DNA oligonucleotides excluding the RNA promoter.

### Claim 2 (depends on 1)

A method according to claim 1, wherein the single RNA polynucleotide is a guide RNA.

### Claim 3 (depends on 1)

A method according to claim 1, further comprising a Cas nuclease capable of being activated in the presence of the guide RNA.

### Claim 4 (depends on 1)

A method according to claim 1, further comprising adding the Cas nuclease in step (a)

### Claim 5 (depends on 1)

A method according to claim 1, wherein the single RNA polynucleotide is selected from the group consisting of a guide RNA, an aptamer, a mRNA, a tRNA, a microRNAs a shRNA, an snRNA, a short non-coding RNA, a long non-coding RNA, an RNA probe, and a ribozyme.

### Claim 6 (depends on 1)

A method according to claim 1, further comprising combining a third synthetic ss DNA oligonucleotide and a fourth synthetic ss DNA oligonucleotide in the reaction chamber, wherein the 5′ end of the third synthetic ss DNA oligonucleotide hybridizes to the 5′ end of the second ss DNA oligonucleotide and the 3′ end of the third ss DNA oligonucleotide hybridizes to the 3′ end of the fourth ss DNA oligonucleotide for forming a single RNA polynucleotide comprising the sequences from first, second, third and fourth oligonucleotide excluding the RNA promoter sequence.

### Claim 7 (depends on 1)

A method according to claim 1, wherein the DNA polymerase is a strand displacing polymerase.

### Claim 8 (depends on 6)

A method according to claim 6, further comprising a DNA ligase in the reaction chamber.

### Claim 9 (depends on 1)

A method according to claim 1, further comprising (c) combining a reactor sequence module in the single RNA molecule with a detector molecule; and causing a detectable signal.

### Claim 10 (depends on 9)

A method according to claim 9, wherein the detector molecule is a third sequence module on the first, second, third or fourth ss DNA oligonucleotide.

### Claim 11 (depends on 10)

A method according to claim 10, wherein the reactor sequence module is a first aptamer.

### Claim 12 (depends on 9)

A method according to claim 9, wherein the detector molecule is a second aptamer or a fluorescent marker for binding the first aptamer.

### Claim 13 (depends on 1)

A method according to claim 1, further comprising immobilizing the 5′ end of the RNA polynucleotide on a solid support.

### Claim 14 (depends on 1)

A method according to claim 1, wherein the 5′ end of the RNA polynucleotide comprises a sequence module containing a modified nucleotide.

### Claim 15 (depends on 14)

A method according to claim 14, wherein the modified nucleotide is biotin or desthiobiotin.

### Claim 16 (depends on 1)

A method according to claim 1, wherein the first ss DNA oligonucleotide contains a sequence module in which the sequence is variable.

### Claim 17 (depends on 1)

A method according to claim 1, wherein the variable sequence module is DNA targeting sequence for a Cas nuclease.

### Claim 18 (depends on 1)

A method according to claim 1, wherein the first synthetic ss DNA oligonucleotide has a variable sequence module and the reaction chamber is one position in an array of reaction chambers so that whereas each reaction chamber contains a first ss DNA oligonucleotide, the variable sequence module differs in substantially each reaction chamber.

### Claim 19 (depends on 18)

A method according to claim 18, wherein the second ss DNA oligonucleotide is optionally the same for each well.

### Claim 20 (depends on 17)

A method according to claim 17, wherein the second ss DNA oligonucleotide comprises a tracrRNA sequence module.

### Claim 21 (depends on 1)

A method according to claim 1, further comprising forming a plurality of different guide RNAs.

### Claim 22 (depends on 1)

A method for making an RNA guided protein, comprising:
(a) selecting a protein for guiding to a nucleic acid target using a guide RNA;
(b) making an RNA according to claim 1;
(c) associating the protein with the RNA from (b) to make an RNA guided protein.

### Claim 23 (depends on 22)

A method according to claim 22, wherein the RNA guided protein is an RNA guided nuclease.