# US 12,091,698 B2 · Compositions and methods for RNA synthesis

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- Number: US 12,091,698 B2
- Document ID: US12091698B2
- Status: Granted
- Kind: B2
- Application number: 18/302,247
- Filed: 2023-04-18
- Published: 2023-09-07
- Granted: 2024-09-17
- Earliest claimed: 2020-10-23
- Assignees: LineaRx, Inc.
- Applicants: LineaRx, Inc.
- Inventors: Brendan Hussey; Lai Him Chung
- Web page: https://patentgrep.com/patent/US12091698B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)}
- C12N 11/06: attached to the carrier via a bridging agent
- C12N 11/14: Enzymes or microbial cells immobilised on or in an inorganic carrier
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- 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}
- C12Y 207/07006: DNA-directed RNA polymerase (2.7.7.6)
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2330/30: chemically synthesised
- 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)}
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Provided herein are compositions, methods, devices, and systems for highly accurate and pure RNA synthesis. Also provided herein are nucleic acid libraries comprising RNAs generated by using devices, compositions and methods disclosed herein.

## Claims

### Claim 1

A composition comprising a modified polypeptide, wherein the modified polypeptide comprises:
a purified T7 RNA polymerase, a functional fragment, or a functional variant thereof; and
a purified nucleic acid binding protein,
wherein the purified T7 RNA polymerase and the purified nucleic acid binding protein are heterologous,
wherein the purified T7 RNA polymerase, the functional fragment, or the functional variant thereof,
and the purified nucleic acid binding protein are linked,
wherein the purified nucleic acid binding protein is an avidin, and
wherein the purified nucleic acid binding protein is linked to the C-terminus or N-terminus of the purified RNA polymerase, the functional fragment, or the functional variant thereof by fusion.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the purified T7 RNA polymerase, the functional fragment, or the functional variant thereof, and/or the purified nucleic acid binding protein are capable of being immobilized on a surface.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the surface comprises a solid surface selected from the group consisting of a magnetic bead, an agarose bead, fused silica, sol-gel, silica polymer, silica monolith, cellulose, agar, acrylamide, a gold bead, and a gel matrix.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the solid surface is a magnetic bead.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the purified T7 RNA polymerase, the functional fragment, or the functional variant thereof, and the purified nucleic acid binding protein are linked via a linker, wherein the linker comprises a peptide linker.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the purified T7 RNA polymerase, the functional fragment, or the functional variant thereof, and the purified nucleic acid binding protein are covalently linked via a linker.

### Claim 7 (depends on 5)

The composition of claim 5, wherein the linker comprises at least 14 amino acid residues in length.

### Claim 8 (depends on 5)

The composition of claim 5, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-12 and SEQ ID NOs: 23-32.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the purified T7 RNA polymerase, the functional fragment, or the functional variant thereof, and the purified nucleic acid binding protein are linked via a second purified nucleic acid binding protein, wherein the second purified nucleic acid binding protein comprises a zinc-finger domain, a leucine zipper, a helix-tum-helix (HTH) motif, a helix-loop-helix (HLH) motif, a winged helix (WH), a winged HTH (WHTH) motif, a high mobility group (HMG)-box, a White-Opaque Regulator 3 (Wor3) domain, an oligonucleotide/oligosaccharide-binding (OB)-fold domain, an immunoglobulin fold, a B3 domain, a Transcription Activator-Like Effector (TALE), a TALE-like protein, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the avidin comprises streptavidin, rhizavidin, or neutravidin.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the avidin is a rhizavidin.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the purified T7 RNA polymerase, the functional fragment thereof, or the functional variant thereof comprises at least one amino acid variation relative to a wild type T7 RNA polymerase.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the purified T7 RNA polymerase, the functional fragment thereof, or the functional variant thereof comprises at least one amino acid variation selected from the group consisting of KI 72L, P266L, H772R, N748X, R756M, Q758X, and E775V, relative to the sequence of SEQ ID NO: 1, wherein the position is determined by alignment with the sequence of SEQ ID NO: 1, and wherein X is any amino acid different from the wild type amino acid.

### Claim 14 (depends on 1)

The composition of claim 1, wherein the purified T7 RNA polymerase, the functional fragment thereof, or the functional variant thereof comprises at least two amino acid variations selected from the group consisting of KI 72L, P266L, H772R, N748X, R756M, Q758X, and E775V, relative to the sequence of SEQ ID NO: 1, wherein the position is determined by alignment with the sequence of SEQ ID NO: 1, and wherein X is any amino acid different from the wild type amino acid.

### Claim 15 (depends on 1)

The composition of claim 1, wherein the purified T7 RNA polymerase, the functional fragment thereof, or the functional variant thereof comprises a sequence that has at least 90% identity to the sequence of SEQ ID NO: 1.